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Drugs of abuse testing in meconium
Clinica Chimica Acta 366 (2006) 101 – 111 www.elsevier.com/locate/clinchim
Review
Drugs of abuse testing in meconium Joey Gareri, Julia Klein, Gideon Koren * Motherisk Program, Division of Clinical Pharmacology/Toxicology, Hospital for Sick Children, Toronto, University of Toronto, Toronto, Ontario, Canada Received 21 December 2004; received in revised form 20 October 2005; accepted 21 October 2005 Available online 17 January 2006
Abstract Prenatal substance abuse is an ongoing concern with significant impact on neonatal health and development across socioeconomic lines. Meconium, passed by neonates during their first post-natal bowel movements, is a matrix unique to the developing fetus and contains a long history of prenatal metabolism. Over the last two decades, the use of meconium as a matrix for assessing prenatal exposure to drugs of abuse has yielded methods exhibiting higher sensitivity, easier collection, and a larger window of detection than traditional matrices. Recently, a method has been developed for the analysis of fatty acid ethyl esters in meconium as a biomarker of fetal alcohol exposure, potentially facilitating the future diagnosis of Fetal Alcohol Spectrum Disorder in situations where gestational alcohol consumption history is unknown. Screening for prenatal exposure to illicit and abused licit drugs in meconium is possible by use of a variety of immunoassay methods with conformational analysis usually occurring by GCMS or LCMS. In spite of increased sample preparation time relative to blood and urine, the long metabolic history, coupled with the ease and wide window of collection of meconium make it the ideal matrix for determining fetal drug exposure. D 2005 Elsevier B.V. All rights reserved. Keywords: Meconium; Gestational; Prenatal; Drugs; Alcohol; Screening
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Introduction. . . . . . . . . . . . . . Meconium . . . . . . . . . . . . . . Cocaine . . . . . . . . . . . . . . . . 3.1. Extraction . . . . . . . . . . . 3.2. Screening/confirmation . . . . Opiates . . . . . . . . . . . . . . . . 4.1. Extraction . . . . . . . . . . . 4.2. Screening/confirmation . . . . Cannabinoids . . . . . . . . . . . . . 5.1. Extraction . . . . . . . . . . . 5.2. Screening/confirmation . . . . Amphetamines/phencyclidine. . . . . 6.1. Extraction . . . . . . . . . . . 6.2. Screening/confirmation . . . . Current trends in illicit drug detection
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* Corresponding author. The Hospital for Sick Children, 555 University Avenue, Toronto, Ontario, Canada, M5G 1X8. Tel.: +1 416 813 5781; fax: +1 416 813 7562. E-mail address: [email protected] (G. Koren). 0009-8981/$ - see front matter D 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.cca.2005.10.028
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Ethanol . . . . . . . . . . . . 8.1. Extraction . . . . . . . 8.2. Screening/confirmation 9. Clinical implications . . . . . 10. Conclusion . . . . . . . . . . References . . . . . . . . . . . . .
J. Gareri et al. / Clinica Chimica Acta 366 (2006) 101 – 111
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1. Introduction Prenatal substance abuse is an ongoing concern, with significant impact on neonatal health and development [1– 11]. Drug abuse in pregnancy exists across socioeconomic lines and has been consistently confirmed in numerous prevalence studies over the last decade [12,13]. Rates of prenatal drug use have been determined by maternal report, urine analysis, blood analysis, and meconium analysis ranging from 3.4– 31% for cocaine, 1– 12% for cannabis, 1.6 –21% for opiates, and 0.1– 4% for ethanol in general and high-risk populations [13 – 17]. Over the last two decades, the use of meconium as a matrix for assessing prenatal drug exposure has yielded a method exhibiting higher sensitivity, easier collection, and a larger window of detection than other traditional matrices [18,19]. In addition to its clinical utility in neonatal intervention and follow-up, the capability of meconium analysis to detect drugs of abuse and their multiple metabolites [20] provides a powerful research tool, assisting in the study of maternal – fetal drug disposition and metabolic capabilities specific to the fetus [21 –23].
2. Meconium Meconium comprises the neonate’s first several bowel movements, identified most commonly by its dark green/ black colour and a lack of odour usually inherent to regular feces. It is a highly complex matrix consisting of water, desquamated epithelial cells from the gastrointestinal tract and skin, lanugo (fine neonatal hair), fatty material from the vernix caseosa, bile acids and salts, cholesterol and sterol precursors, blood group substances, enzymes, mucopolysaccharides, sugars, lipids, proteins, trace metals, various pancreatic and intestestinal secretions, as well as residue of swallowed amniotic fluid [24 – 29]. The timing of formation of meconium has been variably reported from within the first trimester [30] to as late as five months of gestation [31]. The assertion that meconium begins to form at approximately 12 weeks of gestation is likely the most accurate. It is at this time that fetal swallowing of amniotic fluid begins [27,29], and the formation of meconium has been evidenced at this time period by the presence of cocaine found in the meconium of early gestational fetuses [26]. Drugs of abuse are deposited into meconium by deposition from bile or urine via fetal swallowing [27]. Fetal swallowing is thought to be the
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mechanism by which drugs are concentrated in the meconium; as the fetus releases urine into the amniotic fluid, any excreted compounds and metabolites are then swallowed and ultimately deposited into the meconium [26]. Most drugs of abuse are capable of crossing the placenta at rates controlled by their molecular size, ionization state, lipophilicity, and degree of plasma protein or placental tissue binding [28]. Placental transfer of most drugs of abuse takes place primarily by passive diffusion due their small molecular size and high lipophilicity; because of this, placental blood flow may be the most critical limiting factor regarding drug transport to the fetus [32]. Ultimately, fetal exposure is a product of maternal consumption, metabolism and elimination, placental transfer and metabolism, and fetal metabolism [28]. These factors make meconium an optimal matrix for identifying in utero exposure as it is considered to be static once deposited in the fetal intestine [33], a preserved record of the ultimate exposure by the fetus. Urine contamination of meconium occurs when a neonate has been exposed to a compound near-term and evacuates drug-contained urine into a meconium soiled diaper. This phenomenon increases the sensitivity of meconium screening due to the augmentation of drug levels in the specimen. Urine contamination can however interfere with the development of dose – response relationships with regard to the level of drugs present in meconium. Urinedeposited drugs have not undergone the same degree of metabolism as meconium-deposited drugs, altering the expected relationships between drugs and metabolites in meconium. The collection of meconium is non-invasive, making sample collection easy [12] and more successful than urine collection [34]. One major advantage of meconium is a relatively wide window for sample collection. Studies using zinc coproporphyrin (a meconium-specific bile pigment) as a marker have determined that meconium is fully evacuated by 125 h post-natally [24]. Other markers of meconium excretion do not appear in the literature and are not used in the standard practice of meconium analysis. The texture and odour of the sample is used to qualitatively distinguish meconium from post-natally produced feces. The findings attributed to the study of zinc coproporphyrin have correlated well with the practice of meconium collection; while all meconium is evacuated by 125 h post-natally, viable analysis appears to be optimal via collection within 72 h, with the later stages of meconium excretion producing a transitional matrix of meconium and feces. Analysis of
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meconium for cocaine and opiates has demonstrated positive results upon third post-natal day sample collection [35]. While 99% of term infants pass their first formed stool by 48 h, in extremely low birth weight infants, of particular interest in the drug-exposed neonatal population, median age of first stool is 3 days, with 90% of infants passing their first stool by day 12 [36]. This window of opportunity for sample collection far beyond 48 h post-natally is a remarkable advantage of this matrix [37]. In general, it can be assumed that in a general neonatal population, meconium can be reliably collected for drug analysis within the first three post-natal days. Meconium is collected by scraping the contents (0.5 g minimum) of the soiled diaper into a specimen collection container. The thick, viscous nature of the substance makes collection quite easy. Meconium for drug analysis should be stored at 20 -C or 80 -C. For transport to external laboratories for analysis, it is advisable to store at 80 -C until frozen and ship on dry ice.
3. Cocaine Cocaine crosses the placenta via passive diffusion [38]. The metabolism of cocaine has been shown to be highly variable, with little consistency in the relative amounts of cocaine and its metabolites among specimens [5,20]. This fact, in combination with the potential augmentation of cocaine and metabolites in meconium by urine contamination in the diaper, has created difficulty in establishing a genuine dose – response relationship [39,40]. In spite of this, there is solid evidence correlating the frequency, timing, and amount of cocaine use by the mother with the amount of cocaine found in meconium by immunoassay [26]. The concentration of cocaine and metabolites in meconium diminishes rapidly 48 h after birth, with the exception of benzoylecgonine, which remains detectable with relative frequency up to twice as long [33]. Cocaine metabolites exhibit a diverse range of polarities [23] and are not uniformly distributed in meconium [41]. The metabolite of cocaine most commonly determined is the hydrolysis product benzoylecgonine. Low confirmation rates were reported for benzoylecgonine [42] prior to the discovery of several additional metabolites which contributed significantly to immunoreactivity in screening methods [20,23]. Cocaine is almost never found alone in meconium, whereas certain metabolites can exist exclusively within a given sample. Benzoylecgonine and cocaethylene have both shared the distinction of being the sole immunoreactive cocaine metabolite present in meconium samples [33], and the highly polar metabolite m-hydroxybenzoylecgonine is the sole compound present in almost one-fourth of screenedpositive samples [42]. This compound has demonstrated immunoreactivity in EMIT and FPIA assays in both the glucuronidated and hydrolyzed state, and has been quantified at levels up to six-fold higher than benzoylecgonine
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[43]. The relative frequencies of cocaine and cocaine metabolite detection in meconium are listed in Table 1. Different species of cocaine metabolites can also be associated with specific drug use patterns. Cocaethylene, norcocaethylene, and ecgonine ethyl ester are all associated with the concurrent abuse of cocaine and ethanol, while anhydroecgonine and anhydroecgonine methyl ester are pyrolytic products of cocaine indicative of Fcrack_ cocaine use [23]. 3.1. Extraction Certain extraction procedures may exclude specific metabolites due to differences in pH that subsequently affect ionization state [41]. Acid extraction procedures were originally used for immunoassay [35,44], but extraction with methanol was later found to result in much higher sensitivity and lower limit of detection [45]. Inclusive extraction procedures have been developed for GCMS, capable of extracting nine metabolites (extraction efficiencies: 25.1– 95%), and LCMS, extracting fifteen metabolites (extraction efficiencies: 38.9 –59.1%) of cocaine simultaneously for confirmational analysis [20,23]. Variability in recoveries between cocaine and its metabolites is primarily due to the solid-phase portion of the extraction [39]. Mixedphase cartridges consisting of non-polar sorbent interspersed with strong cation exchange resin was found most effective in retaining non-polar and cationic analytes [23,30]. 3.2. Screening/confirmation Radioimmunoassay (RIA), fluorescence polarization immunoassay (FPIA), enzyme multiplied immunoassay Table 1 Adapted from Xia et al., 2000.[23] Compound
Abbrev.
Molecular weight (protonated)
Frequency of detection (N = 21)
Anhydroecgoninea Anhydroecgonine methyl estera Ecgonine Ecgonine methyl ester Ecgonine ethyl esterb Benzoylnorecgonine Benzoylecgonine m-hydroxybenzoylecgonine p-hydroxybenzoylecgonine m-hydroxycocaine p-hydroxycocaine Norcocaine Cocaine Norcocaethyleneb Cocaethyleneb Cocaine-N-oxide
AECG AEME
168.0 182.1
18/21 20/21
ECG EME EEE BN BE m-HOBE p-HOBE m-HOCOC p-HOCOC NC COC NCE CE CNO
186.2 200.3 214.2 276.3 290.3 306.2 306.2 320.2 320.2 290.3 304.2 304.2 318.3 320.2
21/21 20/21 18/21 14/21 20/21 16/21 20/21 13/21 21/21 20/21 20/21 20/21 21/21 12/21
Major metabolites. a Pyrolytic products of cocaine indicative of Fcrack_ use. b Ethanol metabolites.
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technique (EMIT), and enzyme-linked immunosorbent assay (ELISA) are commonly used in initial toxicological screening for cocaine and cocaine metabolites. All methods are capable of achieving a high sensitivity of with cut-offs of 50 ng/g (50 ng/mL for EMIT, 25 ng/mL for RIA) or lower for multiple cocaine-related compounds [37,42,46]. In order to increase specificity (i.e., limit the number of false positive results) the cut-off for positive samples is often set at 100 ng/mg for cocaine or benzoylecgonine. The procedure used for extracting, isolating, and/or purifying cocaine and metabolites from meconium has a much more important effect on the sensitivity of a screening method than the immunoassay procedure itself [25,42]. Gas chromatography/mass spectrometry (GCMS) is capable of simultaneously identifying cross-reacting cocaine metabolites [20,47]. The cross-reactivity of specific cocaine metabolites due to structural similarity and affinity to the antibody complex will vary with the specific immunoassay screening method initially used. Information regarding crossreactivity is generally provided within the respective immunoassay kit. Liquid chromatography/mass spectrometry (LCMS) is also capable of simultaneously detecting several cocaine metabolites, including ecgonine, which in one study was found to be the most prevalent metabolite in meconium [23]. Ecgonine is not normally determined by GCMS due to difficulties in the extraction process [23]. In addition, LCMS does not require the sample derivatization needed for GCMS analysis, making sample preparation one step simpler.
4. Opiates Animal studies have shown that opiates distribute readily across the placenta, resulting in peak fetal blood levels occurring relatively quickly post-intravenous administration as studied with meperidine, methadone and morphine [32]. Morphine distributes widely and variably into many fetal tissues, independent of administered dose [19]. Correlations have been demonstrated between higher dose, longer duration and later gestational timing of exposure and increased levels of morphine in meconium [48]. The extent of fetal exposure to different opiates, based on animal studies, is in the order of meperidine > methadone > morphine [32]. Heroin, based on its high degree of lipid solubility, would carry a high index of fetal exposure as it diffuses readily across the placenta as well as the blood –brain barrier. While this increased fetal exposure has not been specifically shown in animal studies, this mechanism of increased membrane permeability due to the addition of two acetyl groups to the morphine molecule is understood to be the mitigating factor in the pronounced central nervous system effects seen with heroin over the more peripheral activity of morphine. Human studies have shown morphine detectable in meconium collected on the third post-natal day in 75% of subjects upon serial analysis [49]. Higher levels seen in second day-collected samples suggest that hydrolysis of
glucuronidated morphine in meconium and subsequent reabsorption from the intestine occurs [19]. Morphine is extensively glucuronidated in the liver producing the major metabolites morphine-3-glucuronide (inactive), morphine-6glucuronide (active), and normorphine [32]. Bound morphine is found in much higher concentrations in the bile than in other tissues and fluids [19], predicting ready deposition into meconium. Heroin is rapidly deacetylated in vivo to the active metabolite 6-monoacetylmorphine [50]. Fetal liver microsomes are also capable of N-dealkylating meperidine and methadone [32]. The principal metabolite of methadone, 2-ethylidene-1,5-dimethyl-3,3-diphenyl-pyrrolidine has also been reported in meconium [51]. Fetal exposure to opiate is mediated by placental clearance, maternal and fetal protein binding, and fetal microsome activity [32]. While several opiate metabolites have been determined in meconium, the number of studies dedicated to opiate and other drug metabolites is quite small in comparison to the extent of cocaine research; therefore opiate information comparable to that provided for cocaine in Table 1 is not available. 4.1. Extraction Acid and methanolic extractions can be used to extract opiates from meconium with varying degrees of success [25,47,52]. Levels of codeine, hydrocodone, and hydromorphone are known to increase markedly following acid hydrolysis of meconium, indicating a variable degree of glucuronidation in these compounds [25]. Simultaneous methanolic extraction of morphine, codeine, 6-monoacetylmorphine, hydrocodone, and hydromorphone can be carried out with extraction efficiencies between 56– 68% [47]. 4.2. Screening/confirmation Immunoassay analysis for opiates using FPIA and enzyme-based systems can exhibit significant cross-reactivity between illicit and prescribed opiates [47,52]. In spite of this, some radioimmunoassay systems for free morphine analysis have demonstrated negligible cross-reactivity with hydrocodone [53].The immunoreactivity displayed by hydrocodone and its metabolite hydromorphone as well as meperidine and codeine warrants confirmational analysis in cases of opiate-positive screens in order to avoid unnecessary labeling or misdiagnosis. The sensitivities of the opiate immunoassays range from 25 ng/g (FPIA), to 25 ng/mL (RIA), to 100 ng/g (EMIT) [52,54,55]. The cutoff value for positive results is set at 100 ng/g of meconium. GCMS confirmation of opiate-positive immunoassays is capable of determining multiple opiate metabolites, including morphine, codeine, 6-monoacetylmorphine, hydromorphone, and hydrocodone [47,56]. The cut-off of all these analytes is 50 ng/g [47]. Although the assay sensitivity was far below the cutoff levels, these levels were selected to minimize the number of unconfirmed positives. Naturally,
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the cutoff value is higher in the immunoassays than in the confirmatory methods because the immunoassays are specific for the entire group of opiates not for each individual opiate, while GC/MS exhibits very high specificity for each individual opiates.
5. Cannabinoids The plant Cannabis sativa produces over fifty unique compounds known as cannabinoids, with D9-tetrahydrocannabinol (THC) being the most prevalent and psychoactive. Both human and animal studies have shown that cannabinoids cross the placenta, resulting in peak fetal levels after approximately two hours post-inhalation [32]. Levels in the maternal circulation remain higher than those on the fetal side at all times after smoke inhalation, presumably due to extensive protein binding [32,57]. The higher frequency of episodic use of cannabis in pregnancy, compared to other drugs of abuse, limits the sensitivity of meconium analysis in detecting infrequent or isolated fetal cannabinoid exposure [54]. No dose – response relationship or correlations have been established regarding the level of use of marijuana in pregnancy and the levels of cannabinoids found in meconium. Upon serial analysis of meconium for cannabinoids, there is a 60% rate of subsequent detection if the initially collected sample was positive [54]. THC is metabolized to the active compound 11-hydroxyD9-tetrahydrocannabinol (11-OH-THC), which is principally excreted in feces [32]. A subsequent metabolite, 11-norD9-tetrahydrocannabinol-9-carboxylic acid (THC-COOH), is most often used in meconium analysis due to the fact that it is the major urinary metabolite of THC [54,58] and most immunoassays are also used in urinalysis. 8h-hydroxy-D9tetrahydrocannabinol (8h-OH-THC) and 8h,11-dihydroxyD9-tetrahydrocannabinol (8h,11-diOH-THC) have also been identified as THC metabolites found in meconium [59]. 5.1. Extraction Extraction of cannabinoids for immunoassay can be carried out via normal saline (RIA) [34], methanol (EMIT/ ELISA) [46,47], and acetic acid/diphenylamine in acetone (FPIA) [58]. Methanolic extraction of THC-COOH from meconium is capable of yielding a recovery as high as 79% [47]. For GCMS confirmation, an additional hexane/ethyl acetate extraction step is required [47,58]. Base hydrolysis improves the extraction of THC-COOH, indicating that it is significantly glucuronidated in meconium [25,58].
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(EMIT) [47]. ELISA demonstrated a confirmation rate of 100% in one study confirmed by GCMS [46]. THC, 11-OHTHC, 8h-OH-THC, and 8h,11-diOH-THC are all considered major contributors to immunoreactivity when testing for THC-COOH using EMIT [47]. Immunoassay screening for cannabinoids demonstrates sensitivities ranging from 22.7% to 98% [46,54]. This range of sensitivity is likely due to differences in the relative study population as infrequent cannabis users are thought to increase the rate of falsenegatives [54]. The specificity of GCMS confirmational analysis in meconium testing for cannabinoids is dependant on the number of metabolites determined and the extraction procedure utilized. The use of THC-COOH as a primary analyte means that any extraction procedure that reduces the ratio of THC-COOH/total cannabinoids will result in lower rates of confirmation [47]. This can be adjusted by optimizing THC-COOH extraction [46,58] or determining a greater number of potentially immunoreactive cannabinoids and THC metabolites via mass spectrometry [25,47]. GCMS confirmation of THC-COOH can produce a limit of detection as low as 2 ng/g [58].
6. Amphetamines/phencyclidine Amphetamines and phencyclidine are used in pregnancy, although less extensively relative to other drugs of abuse [15,55,60]. Methamphetamine has been shown to cross the placenta within thirty seconds of intraperitoneal injections in animal studies. Peak concentrations are lower on the fetal side, but slower elimination results in prolonged fetal exposure relative to the mother [61]. While no dose – response relationship has been established for amphetamines in meconium, individual case reports show agreement between self-reported heavy prenatal use of methamphetamine and high concentrations (200 – 1000 ng/g) in meconium [60]. Animal studies of phencyclidine demonstrated somewhat slower placental transfer, reaching peak fetal levels 2 h post-parenteral administration. Phencyclidine appears to accumulate in fetal tissue, with fetal levels reaching 10-fold higher than maternal blood [62]. Multiple derivatives and metabolites of amphetamine, including; 3,4-methylenedioxyamphetamine (MDA), 3, 4-methylene-dioxy-methamphetamine (MDMA), 4-hydroxy-3-methoxy-methamphetamine (HMMA), 3,4-methylene-dioxyethyl-amphetamine (MDEA), N-methyl-1-(3, 4-methylene-dioxyphenyl)-2-butanamine (MBDB) are extractable from meconium, but generally only the parent compounds (amphetamine/methamphetamine/MDMA) seem to be detected in practice [60].
5.2. Screening/confirmation 6.1. Extraction RIA, FPIA, EMIT, and ELISA are all used in screening for cannabinoids in meconium. Limits of detection for these methods range from 100 ng/mL (RIA) [34] to 20 ng/g
For screening purpose, amphetamines and phencyclidine can be extracted using methanol. For confirmation by
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GCMS, amphetamines can be extracted from meconium by chloroform in basic conditions followed by derivatization with heptafluorobutyric anhydride. Extraction efficiencies by this method are reported between 40 –55% for amphetamine and methamphetamine [47,55]. Methanol and solidphase extractions for LCMS produced mean recoveries of 61.1% for amphetamine and 87.2% for methamphetamine across a range of concentrations [60]. Phencyclidine extraction has also been reported using ethyl acetate with an extraction efficiency of 21% [47].
high: 80– 100 ng/g for cocaine, benzoylecgonine, opiates and amphetamines and 10 ng/g for PCP and cannabinoids. For confirmation, LCMS is gaining in popularity, due to the fact that there is no need for derivatization and in most cases LCMS has a higher sensitivity than GCMS. These issues become extremely important when a small amount of meconium is available for testing. In many clinical cases there is not enough meconium for both: screening and confirmation and the analyst has to judge if it is more beneficial to perform a broad screening or a more specific test targeted to one specific family of drugs.
6.2. Screening/confirmation Screening for amphetamines and phencyclidine has been described via EMIT immunoassay. This method demonstrates a cut-off of 200 ng/g and 20 ng/g for amphetamines and phencyclidine, respectively [47]. It has been speculated, due to difficulties with confirmation, that unknown metabolites of phencyclidine contribute to immunoreactivity upon meconium screening [25]. Confirmation of amphetamines by LC-MS with electrospray ionization detection exhibits a limit of detection of 1 ng/g for all amphetamine derivatives listed above [60]. GCMS validation is also reported with limits of detection of 50 ng/g, and 3 ng/g for amphetamine/methamphetamine and phencyclidine, respectively [47].
7. Current trends in illicit drug detection ELISA is rapidly becoming the favored method for screening. While the assay sensitivity can be as low as 5 ng/g of meconium when 0.5 g of meconium is used for the analysis, the cutoff for the positive values is still kept rather
8. Ethanol Ethanol is a small molecule that distributes rapidly into total body water and passes readily across the placenta. Ethanol is cleared rapidly from the blood following pseudozero order kinetics [63] resulting in a limited window of detection in blood and urine. The use of multiple maternal markers for alcoholism was found to be ineffective in accurately detecting prenatal alcohol abuse [64], warranting the need for a biomarker specific for pregnancy and directly related to fetal exposure. Fatty acid ethyl esters (FAEE) are non-oxidative ethanol metabolites produced by the esterification of ethanol and free fatty acids, catalyzed primarily by FAEE synthases [65] (see Fig. 1). While the incorporation of fatty acids is tissueand enzyme-dependent, human placenta, animal placenta, heart, and liver are all reported to have significant FAEE synthase activities [66]. Selected FAEE have been recovered from meconium at levels correlating well with maternal selfreported gestational alcohol consumption [67,68], or tolerance for alcohol [69]. Several studies suggest the presence
NON-OXIDATIVE PATHWAY Fatty Acids Ethanol
Fatty Acid Ethyl Esters
FAEE synthases
ADH OXIDATIVE PATHWAY
Acetaldehyde ALDH
Acetic Acid
Deposition in Fatty Tissues and Meconium CO2 + H2O
ADH: alcohol dehydrogenase ALDH: acetaldehyde dehydrogenase Fig. 1. Ethanol metabolism and the formation of fatty acid ethyl esters. ADH: alcohol dehydrogenase, ALDH: acetaldehyde dehydrogenase.
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Table 2 Fatty acid ethyl esters FAEE
Mw
Molecular formula
Details
Ethyl laurate
228.4
C14H28O2
Commonly found in non-exposed population, not included in cumulative calculation [67,70]
Ethyl myristate Ethyl palmitate
256.4 284.5
C16H32O2 C18H36O2
Ethyl Ethyl Ethyl Ethyl Ethyl Ethyl Ethyl
282.5 312.5 310.5 308.5 306.5 332.5 298.5
C18H34O2 C20H40O2 C20H38O2 C20H36O2 C20H34O2 C22H36O2 C19H38O2
palmitoleate stearate oleate linoleate linolenate arachidonate heptadecanoate
of individual FAEE species correlate well with maternal alcohol consumption [67,70], however it has been shown that the total level of multiple common FAEE (as opposed to one selected FAEE) quantified in meconium is a more reliable biomarker by providing a redundancy system for individual variability in fatty acid profiles [68,71]. One case report demonstrated cumulative FAEE levels in a confirmed drinker 34-fold higher than non-drinking controls [68]. The FAEE species of interest in meconium are ethyl laurate, ethyl myristate, ethyl palmitate, ethyl palmitoleate, ethyl stearate, ethyl oleate, ethyl linoleate, ethyl linolenate, and ethyl arachidonate [72].
Cumulatively calculated in the establishment of the 2.0 nmol/g baseline cut-off [67,70]
Used as single biomarkers [67,70]
Internal standard; not endogenously produced
when quantifying FAEE is a more accurate reflection of the amount of esterified ethanol due to the variability in molecular weights exhibited by the various FAEE species. Table 2 provides some descriptive information regarding the individual esters and their roles regarding FAEE calculation and the determination of fetal alcohol exposure. Confirmation of FAEE in meconium can be carried out using the above-mentioned method with a mass spectrometer instead of a flame ionization detector [70,73]. One study confirmed the presence of FAEE above 50 ng/g in 16.7% of a mixed general neonatal and neonatal intensive care unit population [73].
8.1. Extraction 9. Clinical implications FAEE are isolated from meconium via hexane/acetone and solid-phase extraction [71,73]. This method yields extraction efficiencies in our laboratory ranging from 20% (ethyl laurate) to 93% (ethyl arachidonate) for the nine FAEE species, with a mean recovery of approximately 70%. 8.2. Screening/confirmation Since ethanol is also produced endogenously by gut flora [74], the presence of low levels of FAEE in meconium are not necessarily indicative of exogenous ethanol. One study identified cumulative FAEE levels greater than 10,000 ng/g as being indicative of significant alcohol exposure [73]. A baseline study of the meconium of children born to 197 nondrinking women in two distinct populations yielded a positive cut-off of 2.0 nmol cumulative FAEE/g meconium with a sensitivity of 100% and specificity of 98.4% [71]. The longer-chain esters appear to be selectively expressed in alcohol-exposed populations, while the shorter-chain esters are more common in individuals who test below 2.0 nmol/g [71]. The baseline was established using gas chromatography with flame-ionization detection exhibiting a limit of detection of 50 ng/g and a limit of quantitation of approximately 100 ng/g [71]. The use of molar concentrations
The clinical effects of prenatal exposure to cocaine are not conclusive. While some studies have claimed neuroteratogenicity as an adverse outcome related to prenatal exposure [75], later systematic reviews could not detect long-term developmental consequences directly attributable to cocaine [76]. Associated effects such as dose-related decrease in fetal weight and head size [2], prolonged hospitalization [77], and increased early life problems [6] may not be due to cocaine itself, but rather due to a large number of confounders associated with prenatal drug abuse (Table 3). Upon controlling for confounders, only placental Table 3 Confounders in assessing poor perinatal outcome associated with illicit drug use in addicted populations [83,84] Cigarette smoking Concurrent alcohol use Multiple drug use Sexually transmitted disease Poverty Poor prenatal care Poor nutrition (maternal) Poor nutrition (post-natal) Poor social environment (post-natal)
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abruption and premature rupture of membranes were found to be attributable to prenatal cocaine use [1]. Withdrawal manifestations are the most significant concern regarding prenatal exposure to opiates [7,8,40,78]. It has been speculated that reabsorption of morphine from intestinal tissue and meconium may actually alleviate potential opiate withdrawal in addicted neonates [11]. This reabsorption scenario is rarely seen clinically and 67– 90% of heavily exposed neonates exhibit withdrawal symptoms, requiring appropriate therapy ranging from supportive care to pharmacological treatment with opiates or sedatives [79]. The pronounced effects of withdrawal seen in neonates in the absence of large morphine concentrations in CNS tissues indicates a high neonatal sensitivity to morphine as determined by human case reports and studies in nonhuman primates [19]. The signs and symptoms of withdrawal are listed in Fig. 2 [80]. Infrequent use of cannabis in pregnancy may have little to no effect of the fetus [3] however it is not known if there is a threshold level use for adverse developmental effects that is any way related to the screening cut-off values [4]. Unlike other drugs of abuse, acute complications at birth are unlikely to occur via cannabis exposure alone. Prenatal exposure to amphetamines has been linked to oral clefting, cardiac anomalies, and intrauterine growth retardation in both human and animal studies [9]. A comparison of phencyclidine-exposed neonates to matched cocaine-exposed neonates demonstrated a nearly 2-fold increase in incidence of intrauterine growth retardation, pre-term labour, withdrawal/intoxication, and prolonged hospitalization [10]. While confounding factors (see Table 3) are thought to interfere with the definitive establishment of causality in cocaine-exposed neonates, the increased frequency with which these adverse effects are seen in phencyclidine-exposed neonates indicates that prenatal exposure is of significant clinical concern. While the adverse effects of these compounds are significant, the low incidence of prenatal use [15,55,60] dictates that screening is likely beneficial only upon suspicion of use.
It is likely that designer drugs in this class (e.g., MDMA) are used mainly for recreational purposes and discontinued upon realization of pregnancy [81]. Fetal alcohol spectrum disorder (FASD) is a detrimental outcome of maternal alcohol abuse during pregnancy. Clinical presentation of FASD consists of a complex pattern of behavioral or cognitive abnormalities that are inconsistent with the patient’s level of development, familial and environmental background, and/ or evidence of CNS neurodevelopmental abnormalities [16]. A delayed diagnosis may exacerbate the primary insult produced by prenatal alcohol exposure, warranting a neonatal screen in order to increase the capability of intervention. A spectrum of secondary disabilities such as trouble with law, mental health and behavioral problems, and low social adaptability may arise from the primary organic insult and are preventable with early intervention [16,82]. One issue that arises with the use of FAEE as a marker for ethanol exposure is the inability for social service workers to fully comprehend the terminology. While using molar concentrations is necessitated by the variability in molecular weight between FAEE species, the concepts of Fmoles_ and Fethyl esters_ are not well understood by the lay public. An appropriate alternative to expressing results this way would be using the concept of ‘‘bound alcohol’’. Since the ethanol in FAEE is equimolar (i.e., one ethanol molecule per FAEE molecule), a conversion to a mass-based expression similar to those used in drugs of abuse (i.e., ng/g) would apply. The baseline cut-off could then be re-stated as ‘‘92 ng/g of bound alcohol’’.
10. Conclusion While detecting neonatal withdrawal is one objective commonly listed in favour of neonatal drug screening, one study demonstrated that only 10% of screened infants with Fwithdrawal symptoms_ tested positive for drugs of abuse, with 75% of infants with positive screens being asymptom-
Signs and Symptoms of Withdrawal[80] Wakefulness Irritability Tremulousness, Temperature variation, Tachypnea Hyperactivity, High-pitched cry, Hyperacusia, Hyperreflexia, Hypertonus Diarrhea, Diaphoresis, Disorganized suck Rub marks (excoriations of knees and face), Respiratory distress, Rhinorrhea Apneic spells, Autonomic dysfunction Weight loss or failure to gain weight Alkalosis (respiratory) Lacrimation Fig. 2. Signs and symptoms of withdrawal [80].
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atic [37]. In many cases, the benefit of screening lies mainly in the potential for intervention based on the developmentally detrimental lifestyle factors associated with prenatal drug abuse [40]. It is important to note that the use of unvalidated screening data to assess prenatal drug abuse can be unethical and at times dangerous [42]. The massspectrometer is the preferred detector for confirmational analysis due to its unequalled specificity [37]. GCMS and LCMS methods exhibit the ease of multi-sample processing in conjunction with reproducibility and high recovery rates for corresponding extraction procedures [23,33]. The ability of meconium analysis to detect prenatal drug use across the latter two-thirds of pregnancy not only improves sensitivity over alternative screening matrices, but is customized to the detection of addicted behaviour. Since meconium analysis cannot determine drug use in the first trimester, the method effectively protects against the labeling of mothers who ceased their drug or alcohol use upon the detection of pregnancy. In spite of increased sample preparation time relative to blood and urine, the long metabolic history, coupled with the ease and wide window of collection of meconium make it the ideal matrix for determining fetal drug exposure. References [1] Addis A, Moretti ME, Ahmed SF, Einarson TR, Koren G. Fetal effects of cocaine: an updated meta-analysis. Reprod Toxicol 2001;15:341 – 69. [2] Church MW, Morbach CA, Subramanian MG. Comparative effects of prenatal cocaine, alcohol, and undernutrition on maternal/fetal toxicity and fetal body composition in the Sprague – Dawley rat with observations on strain-dependent differences. Neurotoxicol Teratol 1995;17:559 – 67. [3] Fried PA, Watkinson B. 36- and 48-month neurobehavioral follow-up of children prenatally exposed to marijuana, cigarettes, and alcohol. J Dev Behav Pediatr 1990;11:49 – 58. [4] Goldschmidt L, Day NL, Richardson GA. Effects of prenatal marijuana exposure on child behavior problems at age 10. Neurotoxicol Teratol 2000;22:325 – 36. [5] Konkol RJ, Murphey LJ, Ferriero DM, Dempsey DA, Olsen GD. Cocaine metabolites in the neonate: potential for toxicity. J Child Neurol 1994;9:242 – 8. [6] Oro AS, Dixon SD. Perinatal cocaine and methamphetamine exposure: maternal and neonatal correlates. J Pediatr 1987;111:571 – 8. [7] Ostrea EM, Chavez CJ, Strauss ME. A study of factors that influence the severity of neonatal narcotic withdrawal. J Pediatr 1976;88:642 – 5. [8] Ostrea EM, Chavez CJ. Perinatal problems (excluding neonatal withdrawal) in maternal drug addiction: a study of 830 cases. J Pediatr 1979;94:292 – 5. [9] Plessinger MA. Prenatal exposure to amphetamines. Risks and adverse outcomes in pregnancy. Obstet Gynecol Clin North Am 1998;25:119 – 38. [10] Tabor BL, Smith-Wallace T, Yonekura ML. Perinatal outcome associated with PCP versus cocaine use. Am J Drug Alcohol Abuse 1990;16:337 – 48. [11] Zuspan FP, Gumpel JA, Mejia-Zelaya A, Madden J, Davis R. Fetal stress from methadone withdrawal. Am J Obstet Gynecol 1975; 122:43 – 6. [12] Ryan RM, Wagner CL, Schultz JM, et al. Meconium analysis for improved identification of infants exposed to cocaine in utero. J Pediatr 1994;125:435 – 40.
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Review
Drugs of abuse testing in meconium Joey Gareri, Julia Klein, Gideon Koren * Motherisk Program, Division of Clinical Pharmacology/Toxicology, Hospital for Sick Children, Toronto, University of Toronto, Toronto, Ontario, Canada Received 21 December 2004; received in revised form 20 October 2005; accepted 21 October 2005 Available online 17 January 2006
Abstract Prenatal substance abuse is an ongoing concern with significant impact on neonatal health and development across socioeconomic lines. Meconium, passed by neonates during their first post-natal bowel movements, is a matrix unique to the developing fetus and contains a long history of prenatal metabolism. Over the last two decades, the use of meconium as a matrix for assessing prenatal exposure to drugs of abuse has yielded methods exhibiting higher sensitivity, easier collection, and a larger window of detection than traditional matrices. Recently, a method has been developed for the analysis of fatty acid ethyl esters in meconium as a biomarker of fetal alcohol exposure, potentially facilitating the future diagnosis of Fetal Alcohol Spectrum Disorder in situations where gestational alcohol consumption history is unknown. Screening for prenatal exposure to illicit and abused licit drugs in meconium is possible by use of a variety of immunoassay methods with conformational analysis usually occurring by GCMS or LCMS. In spite of increased sample preparation time relative to blood and urine, the long metabolic history, coupled with the ease and wide window of collection of meconium make it the ideal matrix for determining fetal drug exposure. D 2005 Elsevier B.V. All rights reserved. Keywords: Meconium; Gestational; Prenatal; Drugs; Alcohol; Screening
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Introduction. . . . . . . . . . . . . . Meconium . . . . . . . . . . . . . . Cocaine . . . . . . . . . . . . . . . . 3.1. Extraction . . . . . . . . . . . 3.2. Screening/confirmation . . . . Opiates . . . . . . . . . . . . . . . . 4.1. Extraction . . . . . . . . . . . 4.2. Screening/confirmation . . . . Cannabinoids . . . . . . . . . . . . . 5.1. Extraction . . . . . . . . . . . 5.2. Screening/confirmation . . . . Amphetamines/phencyclidine. . . . . 6.1. Extraction . . . . . . . . . . . 6.2. Screening/confirmation . . . . Current trends in illicit drug detection
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* Corresponding author. The Hospital for Sick Children, 555 University Avenue, Toronto, Ontario, Canada, M5G 1X8. Tel.: +1 416 813 5781; fax: +1 416 813 7562. E-mail address: [email protected] (G. Koren). 0009-8981/$ - see front matter D 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.cca.2005.10.028
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Ethanol . . . . . . . . . . . . 8.1. Extraction . . . . . . . 8.2. Screening/confirmation 9. Clinical implications . . . . . 10. Conclusion . . . . . . . . . . References . . . . . . . . . . . . .
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1. Introduction Prenatal substance abuse is an ongoing concern, with significant impact on neonatal health and development [1– 11]. Drug abuse in pregnancy exists across socioeconomic lines and has been consistently confirmed in numerous prevalence studies over the last decade [12,13]. Rates of prenatal drug use have been determined by maternal report, urine analysis, blood analysis, and meconium analysis ranging from 3.4– 31% for cocaine, 1– 12% for cannabis, 1.6 –21% for opiates, and 0.1– 4% for ethanol in general and high-risk populations [13 – 17]. Over the last two decades, the use of meconium as a matrix for assessing prenatal drug exposure has yielded a method exhibiting higher sensitivity, easier collection, and a larger window of detection than other traditional matrices [18,19]. In addition to its clinical utility in neonatal intervention and follow-up, the capability of meconium analysis to detect drugs of abuse and their multiple metabolites [20] provides a powerful research tool, assisting in the study of maternal – fetal drug disposition and metabolic capabilities specific to the fetus [21 –23].
2. Meconium Meconium comprises the neonate’s first several bowel movements, identified most commonly by its dark green/ black colour and a lack of odour usually inherent to regular feces. It is a highly complex matrix consisting of water, desquamated epithelial cells from the gastrointestinal tract and skin, lanugo (fine neonatal hair), fatty material from the vernix caseosa, bile acids and salts, cholesterol and sterol precursors, blood group substances, enzymes, mucopolysaccharides, sugars, lipids, proteins, trace metals, various pancreatic and intestestinal secretions, as well as residue of swallowed amniotic fluid [24 – 29]. The timing of formation of meconium has been variably reported from within the first trimester [30] to as late as five months of gestation [31]. The assertion that meconium begins to form at approximately 12 weeks of gestation is likely the most accurate. It is at this time that fetal swallowing of amniotic fluid begins [27,29], and the formation of meconium has been evidenced at this time period by the presence of cocaine found in the meconium of early gestational fetuses [26]. Drugs of abuse are deposited into meconium by deposition from bile or urine via fetal swallowing [27]. Fetal swallowing is thought to be the
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mechanism by which drugs are concentrated in the meconium; as the fetus releases urine into the amniotic fluid, any excreted compounds and metabolites are then swallowed and ultimately deposited into the meconium [26]. Most drugs of abuse are capable of crossing the placenta at rates controlled by their molecular size, ionization state, lipophilicity, and degree of plasma protein or placental tissue binding [28]. Placental transfer of most drugs of abuse takes place primarily by passive diffusion due their small molecular size and high lipophilicity; because of this, placental blood flow may be the most critical limiting factor regarding drug transport to the fetus [32]. Ultimately, fetal exposure is a product of maternal consumption, metabolism and elimination, placental transfer and metabolism, and fetal metabolism [28]. These factors make meconium an optimal matrix for identifying in utero exposure as it is considered to be static once deposited in the fetal intestine [33], a preserved record of the ultimate exposure by the fetus. Urine contamination of meconium occurs when a neonate has been exposed to a compound near-term and evacuates drug-contained urine into a meconium soiled diaper. This phenomenon increases the sensitivity of meconium screening due to the augmentation of drug levels in the specimen. Urine contamination can however interfere with the development of dose – response relationships with regard to the level of drugs present in meconium. Urinedeposited drugs have not undergone the same degree of metabolism as meconium-deposited drugs, altering the expected relationships between drugs and metabolites in meconium. The collection of meconium is non-invasive, making sample collection easy [12] and more successful than urine collection [34]. One major advantage of meconium is a relatively wide window for sample collection. Studies using zinc coproporphyrin (a meconium-specific bile pigment) as a marker have determined that meconium is fully evacuated by 125 h post-natally [24]. Other markers of meconium excretion do not appear in the literature and are not used in the standard practice of meconium analysis. The texture and odour of the sample is used to qualitatively distinguish meconium from post-natally produced feces. The findings attributed to the study of zinc coproporphyrin have correlated well with the practice of meconium collection; while all meconium is evacuated by 125 h post-natally, viable analysis appears to be optimal via collection within 72 h, with the later stages of meconium excretion producing a transitional matrix of meconium and feces. Analysis of
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meconium for cocaine and opiates has demonstrated positive results upon third post-natal day sample collection [35]. While 99% of term infants pass their first formed stool by 48 h, in extremely low birth weight infants, of particular interest in the drug-exposed neonatal population, median age of first stool is 3 days, with 90% of infants passing their first stool by day 12 [36]. This window of opportunity for sample collection far beyond 48 h post-natally is a remarkable advantage of this matrix [37]. In general, it can be assumed that in a general neonatal population, meconium can be reliably collected for drug analysis within the first three post-natal days. Meconium is collected by scraping the contents (0.5 g minimum) of the soiled diaper into a specimen collection container. The thick, viscous nature of the substance makes collection quite easy. Meconium for drug analysis should be stored at 20 -C or 80 -C. For transport to external laboratories for analysis, it is advisable to store at 80 -C until frozen and ship on dry ice.
3. Cocaine Cocaine crosses the placenta via passive diffusion [38]. The metabolism of cocaine has been shown to be highly variable, with little consistency in the relative amounts of cocaine and its metabolites among specimens [5,20]. This fact, in combination with the potential augmentation of cocaine and metabolites in meconium by urine contamination in the diaper, has created difficulty in establishing a genuine dose – response relationship [39,40]. In spite of this, there is solid evidence correlating the frequency, timing, and amount of cocaine use by the mother with the amount of cocaine found in meconium by immunoassay [26]. The concentration of cocaine and metabolites in meconium diminishes rapidly 48 h after birth, with the exception of benzoylecgonine, which remains detectable with relative frequency up to twice as long [33]. Cocaine metabolites exhibit a diverse range of polarities [23] and are not uniformly distributed in meconium [41]. The metabolite of cocaine most commonly determined is the hydrolysis product benzoylecgonine. Low confirmation rates were reported for benzoylecgonine [42] prior to the discovery of several additional metabolites which contributed significantly to immunoreactivity in screening methods [20,23]. Cocaine is almost never found alone in meconium, whereas certain metabolites can exist exclusively within a given sample. Benzoylecgonine and cocaethylene have both shared the distinction of being the sole immunoreactive cocaine metabolite present in meconium samples [33], and the highly polar metabolite m-hydroxybenzoylecgonine is the sole compound present in almost one-fourth of screenedpositive samples [42]. This compound has demonstrated immunoreactivity in EMIT and FPIA assays in both the glucuronidated and hydrolyzed state, and has been quantified at levels up to six-fold higher than benzoylecgonine
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[43]. The relative frequencies of cocaine and cocaine metabolite detection in meconium are listed in Table 1. Different species of cocaine metabolites can also be associated with specific drug use patterns. Cocaethylene, norcocaethylene, and ecgonine ethyl ester are all associated with the concurrent abuse of cocaine and ethanol, while anhydroecgonine and anhydroecgonine methyl ester are pyrolytic products of cocaine indicative of Fcrack_ cocaine use [23]. 3.1. Extraction Certain extraction procedures may exclude specific metabolites due to differences in pH that subsequently affect ionization state [41]. Acid extraction procedures were originally used for immunoassay [35,44], but extraction with methanol was later found to result in much higher sensitivity and lower limit of detection [45]. Inclusive extraction procedures have been developed for GCMS, capable of extracting nine metabolites (extraction efficiencies: 25.1– 95%), and LCMS, extracting fifteen metabolites (extraction efficiencies: 38.9 –59.1%) of cocaine simultaneously for confirmational analysis [20,23]. Variability in recoveries between cocaine and its metabolites is primarily due to the solid-phase portion of the extraction [39]. Mixedphase cartridges consisting of non-polar sorbent interspersed with strong cation exchange resin was found most effective in retaining non-polar and cationic analytes [23,30]. 3.2. Screening/confirmation Radioimmunoassay (RIA), fluorescence polarization immunoassay (FPIA), enzyme multiplied immunoassay Table 1 Adapted from Xia et al., 2000.[23] Compound
Abbrev.
Molecular weight (protonated)
Frequency of detection (N = 21)
Anhydroecgoninea Anhydroecgonine methyl estera Ecgonine Ecgonine methyl ester Ecgonine ethyl esterb Benzoylnorecgonine Benzoylecgonine m-hydroxybenzoylecgonine p-hydroxybenzoylecgonine m-hydroxycocaine p-hydroxycocaine Norcocaine Cocaine Norcocaethyleneb Cocaethyleneb Cocaine-N-oxide
AECG AEME
168.0 182.1
18/21 20/21
ECG EME EEE BN BE m-HOBE p-HOBE m-HOCOC p-HOCOC NC COC NCE CE CNO
186.2 200.3 214.2 276.3 290.3 306.2 306.2 320.2 320.2 290.3 304.2 304.2 318.3 320.2
21/21 20/21 18/21 14/21 20/21 16/21 20/21 13/21 21/21 20/21 20/21 20/21 21/21 12/21
Major metabolites. a Pyrolytic products of cocaine indicative of Fcrack_ use. b Ethanol metabolites.
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technique (EMIT), and enzyme-linked immunosorbent assay (ELISA) are commonly used in initial toxicological screening for cocaine and cocaine metabolites. All methods are capable of achieving a high sensitivity of with cut-offs of 50 ng/g (50 ng/mL for EMIT, 25 ng/mL for RIA) or lower for multiple cocaine-related compounds [37,42,46]. In order to increase specificity (i.e., limit the number of false positive results) the cut-off for positive samples is often set at 100 ng/mg for cocaine or benzoylecgonine. The procedure used for extracting, isolating, and/or purifying cocaine and metabolites from meconium has a much more important effect on the sensitivity of a screening method than the immunoassay procedure itself [25,42]. Gas chromatography/mass spectrometry (GCMS) is capable of simultaneously identifying cross-reacting cocaine metabolites [20,47]. The cross-reactivity of specific cocaine metabolites due to structural similarity and affinity to the antibody complex will vary with the specific immunoassay screening method initially used. Information regarding crossreactivity is generally provided within the respective immunoassay kit. Liquid chromatography/mass spectrometry (LCMS) is also capable of simultaneously detecting several cocaine metabolites, including ecgonine, which in one study was found to be the most prevalent metabolite in meconium [23]. Ecgonine is not normally determined by GCMS due to difficulties in the extraction process [23]. In addition, LCMS does not require the sample derivatization needed for GCMS analysis, making sample preparation one step simpler.
4. Opiates Animal studies have shown that opiates distribute readily across the placenta, resulting in peak fetal blood levels occurring relatively quickly post-intravenous administration as studied with meperidine, methadone and morphine [32]. Morphine distributes widely and variably into many fetal tissues, independent of administered dose [19]. Correlations have been demonstrated between higher dose, longer duration and later gestational timing of exposure and increased levels of morphine in meconium [48]. The extent of fetal exposure to different opiates, based on animal studies, is in the order of meperidine > methadone > morphine [32]. Heroin, based on its high degree of lipid solubility, would carry a high index of fetal exposure as it diffuses readily across the placenta as well as the blood –brain barrier. While this increased fetal exposure has not been specifically shown in animal studies, this mechanism of increased membrane permeability due to the addition of two acetyl groups to the morphine molecule is understood to be the mitigating factor in the pronounced central nervous system effects seen with heroin over the more peripheral activity of morphine. Human studies have shown morphine detectable in meconium collected on the third post-natal day in 75% of subjects upon serial analysis [49]. Higher levels seen in second day-collected samples suggest that hydrolysis of
glucuronidated morphine in meconium and subsequent reabsorption from the intestine occurs [19]. Morphine is extensively glucuronidated in the liver producing the major metabolites morphine-3-glucuronide (inactive), morphine-6glucuronide (active), and normorphine [32]. Bound morphine is found in much higher concentrations in the bile than in other tissues and fluids [19], predicting ready deposition into meconium. Heroin is rapidly deacetylated in vivo to the active metabolite 6-monoacetylmorphine [50]. Fetal liver microsomes are also capable of N-dealkylating meperidine and methadone [32]. The principal metabolite of methadone, 2-ethylidene-1,5-dimethyl-3,3-diphenyl-pyrrolidine has also been reported in meconium [51]. Fetal exposure to opiate is mediated by placental clearance, maternal and fetal protein binding, and fetal microsome activity [32]. While several opiate metabolites have been determined in meconium, the number of studies dedicated to opiate and other drug metabolites is quite small in comparison to the extent of cocaine research; therefore opiate information comparable to that provided for cocaine in Table 1 is not available. 4.1. Extraction Acid and methanolic extractions can be used to extract opiates from meconium with varying degrees of success [25,47,52]. Levels of codeine, hydrocodone, and hydromorphone are known to increase markedly following acid hydrolysis of meconium, indicating a variable degree of glucuronidation in these compounds [25]. Simultaneous methanolic extraction of morphine, codeine, 6-monoacetylmorphine, hydrocodone, and hydromorphone can be carried out with extraction efficiencies between 56– 68% [47]. 4.2. Screening/confirmation Immunoassay analysis for opiates using FPIA and enzyme-based systems can exhibit significant cross-reactivity between illicit and prescribed opiates [47,52]. In spite of this, some radioimmunoassay systems for free morphine analysis have demonstrated negligible cross-reactivity with hydrocodone [53].The immunoreactivity displayed by hydrocodone and its metabolite hydromorphone as well as meperidine and codeine warrants confirmational analysis in cases of opiate-positive screens in order to avoid unnecessary labeling or misdiagnosis. The sensitivities of the opiate immunoassays range from 25 ng/g (FPIA), to 25 ng/mL (RIA), to 100 ng/g (EMIT) [52,54,55]. The cutoff value for positive results is set at 100 ng/g of meconium. GCMS confirmation of opiate-positive immunoassays is capable of determining multiple opiate metabolites, including morphine, codeine, 6-monoacetylmorphine, hydromorphone, and hydrocodone [47,56]. The cut-off of all these analytes is 50 ng/g [47]. Although the assay sensitivity was far below the cutoff levels, these levels were selected to minimize the number of unconfirmed positives. Naturally,
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the cutoff value is higher in the immunoassays than in the confirmatory methods because the immunoassays are specific for the entire group of opiates not for each individual opiate, while GC/MS exhibits very high specificity for each individual opiates.
5. Cannabinoids The plant Cannabis sativa produces over fifty unique compounds known as cannabinoids, with D9-tetrahydrocannabinol (THC) being the most prevalent and psychoactive. Both human and animal studies have shown that cannabinoids cross the placenta, resulting in peak fetal levels after approximately two hours post-inhalation [32]. Levels in the maternal circulation remain higher than those on the fetal side at all times after smoke inhalation, presumably due to extensive protein binding [32,57]. The higher frequency of episodic use of cannabis in pregnancy, compared to other drugs of abuse, limits the sensitivity of meconium analysis in detecting infrequent or isolated fetal cannabinoid exposure [54]. No dose – response relationship or correlations have been established regarding the level of use of marijuana in pregnancy and the levels of cannabinoids found in meconium. Upon serial analysis of meconium for cannabinoids, there is a 60% rate of subsequent detection if the initially collected sample was positive [54]. THC is metabolized to the active compound 11-hydroxyD9-tetrahydrocannabinol (11-OH-THC), which is principally excreted in feces [32]. A subsequent metabolite, 11-norD9-tetrahydrocannabinol-9-carboxylic acid (THC-COOH), is most often used in meconium analysis due to the fact that it is the major urinary metabolite of THC [54,58] and most immunoassays are also used in urinalysis. 8h-hydroxy-D9tetrahydrocannabinol (8h-OH-THC) and 8h,11-dihydroxyD9-tetrahydrocannabinol (8h,11-diOH-THC) have also been identified as THC metabolites found in meconium [59]. 5.1. Extraction Extraction of cannabinoids for immunoassay can be carried out via normal saline (RIA) [34], methanol (EMIT/ ELISA) [46,47], and acetic acid/diphenylamine in acetone (FPIA) [58]. Methanolic extraction of THC-COOH from meconium is capable of yielding a recovery as high as 79% [47]. For GCMS confirmation, an additional hexane/ethyl acetate extraction step is required [47,58]. Base hydrolysis improves the extraction of THC-COOH, indicating that it is significantly glucuronidated in meconium [25,58].
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(EMIT) [47]. ELISA demonstrated a confirmation rate of 100% in one study confirmed by GCMS [46]. THC, 11-OHTHC, 8h-OH-THC, and 8h,11-diOH-THC are all considered major contributors to immunoreactivity when testing for THC-COOH using EMIT [47]. Immunoassay screening for cannabinoids demonstrates sensitivities ranging from 22.7% to 98% [46,54]. This range of sensitivity is likely due to differences in the relative study population as infrequent cannabis users are thought to increase the rate of falsenegatives [54]. The specificity of GCMS confirmational analysis in meconium testing for cannabinoids is dependant on the number of metabolites determined and the extraction procedure utilized. The use of THC-COOH as a primary analyte means that any extraction procedure that reduces the ratio of THC-COOH/total cannabinoids will result in lower rates of confirmation [47]. This can be adjusted by optimizing THC-COOH extraction [46,58] or determining a greater number of potentially immunoreactive cannabinoids and THC metabolites via mass spectrometry [25,47]. GCMS confirmation of THC-COOH can produce a limit of detection as low as 2 ng/g [58].
6. Amphetamines/phencyclidine Amphetamines and phencyclidine are used in pregnancy, although less extensively relative to other drugs of abuse [15,55,60]. Methamphetamine has been shown to cross the placenta within thirty seconds of intraperitoneal injections in animal studies. Peak concentrations are lower on the fetal side, but slower elimination results in prolonged fetal exposure relative to the mother [61]. While no dose – response relationship has been established for amphetamines in meconium, individual case reports show agreement between self-reported heavy prenatal use of methamphetamine and high concentrations (200 – 1000 ng/g) in meconium [60]. Animal studies of phencyclidine demonstrated somewhat slower placental transfer, reaching peak fetal levels 2 h post-parenteral administration. Phencyclidine appears to accumulate in fetal tissue, with fetal levels reaching 10-fold higher than maternal blood [62]. Multiple derivatives and metabolites of amphetamine, including; 3,4-methylenedioxyamphetamine (MDA), 3, 4-methylene-dioxy-methamphetamine (MDMA), 4-hydroxy-3-methoxy-methamphetamine (HMMA), 3,4-methylene-dioxyethyl-amphetamine (MDEA), N-methyl-1-(3, 4-methylene-dioxyphenyl)-2-butanamine (MBDB) are extractable from meconium, but generally only the parent compounds (amphetamine/methamphetamine/MDMA) seem to be detected in practice [60].
5.2. Screening/confirmation 6.1. Extraction RIA, FPIA, EMIT, and ELISA are all used in screening for cannabinoids in meconium. Limits of detection for these methods range from 100 ng/mL (RIA) [34] to 20 ng/g
For screening purpose, amphetamines and phencyclidine can be extracted using methanol. For confirmation by
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GCMS, amphetamines can be extracted from meconium by chloroform in basic conditions followed by derivatization with heptafluorobutyric anhydride. Extraction efficiencies by this method are reported between 40 –55% for amphetamine and methamphetamine [47,55]. Methanol and solidphase extractions for LCMS produced mean recoveries of 61.1% for amphetamine and 87.2% for methamphetamine across a range of concentrations [60]. Phencyclidine extraction has also been reported using ethyl acetate with an extraction efficiency of 21% [47].
high: 80– 100 ng/g for cocaine, benzoylecgonine, opiates and amphetamines and 10 ng/g for PCP and cannabinoids. For confirmation, LCMS is gaining in popularity, due to the fact that there is no need for derivatization and in most cases LCMS has a higher sensitivity than GCMS. These issues become extremely important when a small amount of meconium is available for testing. In many clinical cases there is not enough meconium for both: screening and confirmation and the analyst has to judge if it is more beneficial to perform a broad screening or a more specific test targeted to one specific family of drugs.
6.2. Screening/confirmation Screening for amphetamines and phencyclidine has been described via EMIT immunoassay. This method demonstrates a cut-off of 200 ng/g and 20 ng/g for amphetamines and phencyclidine, respectively [47]. It has been speculated, due to difficulties with confirmation, that unknown metabolites of phencyclidine contribute to immunoreactivity upon meconium screening [25]. Confirmation of amphetamines by LC-MS with electrospray ionization detection exhibits a limit of detection of 1 ng/g for all amphetamine derivatives listed above [60]. GCMS validation is also reported with limits of detection of 50 ng/g, and 3 ng/g for amphetamine/methamphetamine and phencyclidine, respectively [47].
7. Current trends in illicit drug detection ELISA is rapidly becoming the favored method for screening. While the assay sensitivity can be as low as 5 ng/g of meconium when 0.5 g of meconium is used for the analysis, the cutoff for the positive values is still kept rather
8. Ethanol Ethanol is a small molecule that distributes rapidly into total body water and passes readily across the placenta. Ethanol is cleared rapidly from the blood following pseudozero order kinetics [63] resulting in a limited window of detection in blood and urine. The use of multiple maternal markers for alcoholism was found to be ineffective in accurately detecting prenatal alcohol abuse [64], warranting the need for a biomarker specific for pregnancy and directly related to fetal exposure. Fatty acid ethyl esters (FAEE) are non-oxidative ethanol metabolites produced by the esterification of ethanol and free fatty acids, catalyzed primarily by FAEE synthases [65] (see Fig. 1). While the incorporation of fatty acids is tissueand enzyme-dependent, human placenta, animal placenta, heart, and liver are all reported to have significant FAEE synthase activities [66]. Selected FAEE have been recovered from meconium at levels correlating well with maternal selfreported gestational alcohol consumption [67,68], or tolerance for alcohol [69]. Several studies suggest the presence
NON-OXIDATIVE PATHWAY Fatty Acids Ethanol
Fatty Acid Ethyl Esters
FAEE synthases
ADH OXIDATIVE PATHWAY
Acetaldehyde ALDH
Acetic Acid
Deposition in Fatty Tissues and Meconium CO2 + H2O
ADH: alcohol dehydrogenase ALDH: acetaldehyde dehydrogenase Fig. 1. Ethanol metabolism and the formation of fatty acid ethyl esters. ADH: alcohol dehydrogenase, ALDH: acetaldehyde dehydrogenase.
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Table 2 Fatty acid ethyl esters FAEE
Mw
Molecular formula
Details
Ethyl laurate
228.4
C14H28O2
Commonly found in non-exposed population, not included in cumulative calculation [67,70]
Ethyl myristate Ethyl palmitate
256.4 284.5
C16H32O2 C18H36O2
Ethyl Ethyl Ethyl Ethyl Ethyl Ethyl Ethyl
282.5 312.5 310.5 308.5 306.5 332.5 298.5
C18H34O2 C20H40O2 C20H38O2 C20H36O2 C20H34O2 C22H36O2 C19H38O2
palmitoleate stearate oleate linoleate linolenate arachidonate heptadecanoate
of individual FAEE species correlate well with maternal alcohol consumption [67,70], however it has been shown that the total level of multiple common FAEE (as opposed to one selected FAEE) quantified in meconium is a more reliable biomarker by providing a redundancy system for individual variability in fatty acid profiles [68,71]. One case report demonstrated cumulative FAEE levels in a confirmed drinker 34-fold higher than non-drinking controls [68]. The FAEE species of interest in meconium are ethyl laurate, ethyl myristate, ethyl palmitate, ethyl palmitoleate, ethyl stearate, ethyl oleate, ethyl linoleate, ethyl linolenate, and ethyl arachidonate [72].
Cumulatively calculated in the establishment of the 2.0 nmol/g baseline cut-off [67,70]
Used as single biomarkers [67,70]
Internal standard; not endogenously produced
when quantifying FAEE is a more accurate reflection of the amount of esterified ethanol due to the variability in molecular weights exhibited by the various FAEE species. Table 2 provides some descriptive information regarding the individual esters and their roles regarding FAEE calculation and the determination of fetal alcohol exposure. Confirmation of FAEE in meconium can be carried out using the above-mentioned method with a mass spectrometer instead of a flame ionization detector [70,73]. One study confirmed the presence of FAEE above 50 ng/g in 16.7% of a mixed general neonatal and neonatal intensive care unit population [73].
8.1. Extraction 9. Clinical implications FAEE are isolated from meconium via hexane/acetone and solid-phase extraction [71,73]. This method yields extraction efficiencies in our laboratory ranging from 20% (ethyl laurate) to 93% (ethyl arachidonate) for the nine FAEE species, with a mean recovery of approximately 70%. 8.2. Screening/confirmation Since ethanol is also produced endogenously by gut flora [74], the presence of low levels of FAEE in meconium are not necessarily indicative of exogenous ethanol. One study identified cumulative FAEE levels greater than 10,000 ng/g as being indicative of significant alcohol exposure [73]. A baseline study of the meconium of children born to 197 nondrinking women in two distinct populations yielded a positive cut-off of 2.0 nmol cumulative FAEE/g meconium with a sensitivity of 100% and specificity of 98.4% [71]. The longer-chain esters appear to be selectively expressed in alcohol-exposed populations, while the shorter-chain esters are more common in individuals who test below 2.0 nmol/g [71]. The baseline was established using gas chromatography with flame-ionization detection exhibiting a limit of detection of 50 ng/g and a limit of quantitation of approximately 100 ng/g [71]. The use of molar concentrations
The clinical effects of prenatal exposure to cocaine are not conclusive. While some studies have claimed neuroteratogenicity as an adverse outcome related to prenatal exposure [75], later systematic reviews could not detect long-term developmental consequences directly attributable to cocaine [76]. Associated effects such as dose-related decrease in fetal weight and head size [2], prolonged hospitalization [77], and increased early life problems [6] may not be due to cocaine itself, but rather due to a large number of confounders associated with prenatal drug abuse (Table 3). Upon controlling for confounders, only placental Table 3 Confounders in assessing poor perinatal outcome associated with illicit drug use in addicted populations [83,84] Cigarette smoking Concurrent alcohol use Multiple drug use Sexually transmitted disease Poverty Poor prenatal care Poor nutrition (maternal) Poor nutrition (post-natal) Poor social environment (post-natal)
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abruption and premature rupture of membranes were found to be attributable to prenatal cocaine use [1]. Withdrawal manifestations are the most significant concern regarding prenatal exposure to opiates [7,8,40,78]. It has been speculated that reabsorption of morphine from intestinal tissue and meconium may actually alleviate potential opiate withdrawal in addicted neonates [11]. This reabsorption scenario is rarely seen clinically and 67– 90% of heavily exposed neonates exhibit withdrawal symptoms, requiring appropriate therapy ranging from supportive care to pharmacological treatment with opiates or sedatives [79]. The pronounced effects of withdrawal seen in neonates in the absence of large morphine concentrations in CNS tissues indicates a high neonatal sensitivity to morphine as determined by human case reports and studies in nonhuman primates [19]. The signs and symptoms of withdrawal are listed in Fig. 2 [80]. Infrequent use of cannabis in pregnancy may have little to no effect of the fetus [3] however it is not known if there is a threshold level use for adverse developmental effects that is any way related to the screening cut-off values [4]. Unlike other drugs of abuse, acute complications at birth are unlikely to occur via cannabis exposure alone. Prenatal exposure to amphetamines has been linked to oral clefting, cardiac anomalies, and intrauterine growth retardation in both human and animal studies [9]. A comparison of phencyclidine-exposed neonates to matched cocaine-exposed neonates demonstrated a nearly 2-fold increase in incidence of intrauterine growth retardation, pre-term labour, withdrawal/intoxication, and prolonged hospitalization [10]. While confounding factors (see Table 3) are thought to interfere with the definitive establishment of causality in cocaine-exposed neonates, the increased frequency with which these adverse effects are seen in phencyclidine-exposed neonates indicates that prenatal exposure is of significant clinical concern. While the adverse effects of these compounds are significant, the low incidence of prenatal use [15,55,60] dictates that screening is likely beneficial only upon suspicion of use.
It is likely that designer drugs in this class (e.g., MDMA) are used mainly for recreational purposes and discontinued upon realization of pregnancy [81]. Fetal alcohol spectrum disorder (FASD) is a detrimental outcome of maternal alcohol abuse during pregnancy. Clinical presentation of FASD consists of a complex pattern of behavioral or cognitive abnormalities that are inconsistent with the patient’s level of development, familial and environmental background, and/ or evidence of CNS neurodevelopmental abnormalities [16]. A delayed diagnosis may exacerbate the primary insult produced by prenatal alcohol exposure, warranting a neonatal screen in order to increase the capability of intervention. A spectrum of secondary disabilities such as trouble with law, mental health and behavioral problems, and low social adaptability may arise from the primary organic insult and are preventable with early intervention [16,82]. One issue that arises with the use of FAEE as a marker for ethanol exposure is the inability for social service workers to fully comprehend the terminology. While using molar concentrations is necessitated by the variability in molecular weight between FAEE species, the concepts of Fmoles_ and Fethyl esters_ are not well understood by the lay public. An appropriate alternative to expressing results this way would be using the concept of ‘‘bound alcohol’’. Since the ethanol in FAEE is equimolar (i.e., one ethanol molecule per FAEE molecule), a conversion to a mass-based expression similar to those used in drugs of abuse (i.e., ng/g) would apply. The baseline cut-off could then be re-stated as ‘‘92 ng/g of bound alcohol’’.
10. Conclusion While detecting neonatal withdrawal is one objective commonly listed in favour of neonatal drug screening, one study demonstrated that only 10% of screened infants with Fwithdrawal symptoms_ tested positive for drugs of abuse, with 75% of infants with positive screens being asymptom-
Signs and Symptoms of Withdrawal[80] Wakefulness Irritability Tremulousness, Temperature variation, Tachypnea Hyperactivity, High-pitched cry, Hyperacusia, Hyperreflexia, Hypertonus Diarrhea, Diaphoresis, Disorganized suck Rub marks (excoriations of knees and face), Respiratory distress, Rhinorrhea Apneic spells, Autonomic dysfunction Weight loss or failure to gain weight Alkalosis (respiratory) Lacrimation Fig. 2. Signs and symptoms of withdrawal [80].
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