Oscar Garcia-Algar

Biological matrices for the evaluation of in utero exposure to drugs of abuse.

In recent years, the evaluation of in utero exposure to drugs of abuse has been achieved by testing biological matrices coming from the fetus or newborn (eg, meconium, fetal hair, cord blood, neonatal urine), the pregnant or nursing mother (eg, hair, blood, oral fluid, sweat, urine, breast milk), or from both the fetus and the mother (placenta, amniotic fluid). Overall, these matrices have the advantage of noninvasive collection (with the exception of amniotic fluid) and early detection of exposure from different gestational periods. Matrices such as amniotic fluid, meconium, fetal hair, and maternal hair provide a long historical record of prenatal exposure to certain drugs and can account for different periods of gestation: amniotic fluid from the early pregnancy, meconium for the second and third trimester of gestation, fetal hair for the third, and finally maternal hair (when long enough) for the whole pregnancy. Placenta may reveal the passage of a substance from the mother to the fetus. Cord blood and neonatal urine are useful for determining acute exposure to drugs of abuse in the period immediately previous to delivery. Drug detection in maternal blood, oral fluid, and sweat accounts only for acute consumption that occurred in the hours previous to collection and gives poor information concerning fetal exposure. Different immunoassays were used as screening methods for drug testing in the above-reported matrices or as unique analytical investigation tools when chromatographic techniques coupled to mass spectrometry were not commonly available. However, in the last decade, both liquid and gas chromatography-mass spectrometric methodologies have been routinely applied after appropriate extraction of drugs and their metabolites from these biological matrices.

Determination of maternal-fetal biomarkers of prenatal exposure to ethanol: A review

The deleterious effects exerted by prenatal ethanol exposure include physical, mental, behavioural and/or learning disabilities that are included in the term fetal alcohol spectrum disorder (FASD). Objective assessment of exposure to ethanol at both prenatal and postnatal stages is essential for early prevention and intervention. Since pregnant women tend to underreport alcohol drinking by questionnaires, a number of biological markers have been proposed and evaluated for their capability to highlight gestational drinking behaviour. These biomarkers include classical biomarkers (albeit indirect) of alcohol-induced pathology (mean corpuscular volume (MCV), gamma glutamyltransferase (GGT), aspartate aminotransferase (AST) and alanine aminotransferase (ALT)) acetaldehyde-derived conjugates, and finally derivatives of non-oxidative ethanol metabolism (fatty acid ethyl esters (FAEEs), ethyl glucuronide (EtG), ethyl sulphate (EtS) and phosphaditylethanol (PEth)). Since ethanol itself and acetaldehyde are only measured few hours after ethanol intake in conventional matrices such as blood, urine and sweat, they are only useful to detect recent ethanol exposure. In the past few years, the non-oxidative ethanol metabolites have received increasing attention because of their specificity and in some case wide time-window of detection in non-conventional matrices from the pregnant mother (oral fluid and hair) and fetus-newborn (neonatal hair, meconium, placenta and umbilical cord). This article reviews bioanalytical procedures for the determination of these markers of ethanol consumption during pregnancy and related prenatal exposure. In addition, clinical toxicological applications of these procedures are presented and discussed.

Alarming Prevalence of Fetal Alcohol Exposure in a Mediterranean City

The prevalence of gestational ethanol exposure and subsequent fetal exposure has been assessed in a cohort of mother-infant dyads in a Mediterranean city (Barcelona, Spain) by meconium analysis of fatty acid ethyl esters (FAEEs) after showing in this population a high prevalence of meconium opiates (8.7%), cocaine (4.4%), and cannabis (5.3%). Of the 353 meconium samples analyzed for FAEEs, 159 (45%) contained a total amount of seven FAEEs equal or above 2 nmol/g meconium, the cutoff internationally accepted to differentiate heavy maternal alcohol consumption during pregnancy from occasional use or no use at all. No parental sociodemographic differences or maternal features differentiated exposed from unexposed newborns. The prevalence of gestational consumption of ethanol was similar between women using and not using drugs of abuse during pregnancy (45.7% and 44.7% of samples with total FAEEs equal or higher than 2 nmol/g meconium, respectively). Meconium samples from newborns exposed in utero to ethanol, and positive for at least one illicit drug (cocaine, opiates, or cannabis), had total FAEEs and five of nine individual FAEEs statistically higher than the meconium samples that were negative for the most frequently used illicit drugs of abuse. Among the most prevalent FAEEs, oleic acid ethyl ester showed the best correlation to total FAEE concentration followed by palmitoleic acid ethyl ester. This study, which highlights a 45% ethanol consumption during pregnancy in a low socioeconomic status cohort, may serve as an eye opener for Europeans that gestational alcohol exposure is not endemic only in areas outside of Europe.

Simultaneous analysis of frequently used licit and illicit psychoactive drugs in breast milk by liquid chromatography tandem mass spectrometry

A liquid chromatography tandem mass spectrometry (LC-MS-MS) method for the quantification of frequently used licit (caffeine, nicotine and cotinine) and illicit drugs (opiates, cocaine, cannabinoids and amphetamines) in breast milk was developed and fully validated. Chromatography was performed on a reverse-phase column using a gradient of 2mM ammonium acetate, pH 6.6, and methyl alcohol as mobile phase at a flow rate of 0.35 mL/min. Separated analytes were quantified by electrospray ionization tandem mass spectrometry in positive ion mode using multiple reaction monitoring. Milk samples were kept at -20 °C until analysis and the compounds under investigation were extracted from the matrix by Bond Elut Certify cartridges. The concentration range covered was LOQ to 1000 ng/mL for all the investigated drugs. Intra- and inter-assay imprecision was less than 20%, analytical recovery ranged between 51.6% and 86.5%, matrix effect between 71.1% and 116.6% and process efficiency between 46.8% and 84.0%. Analytes were stable after three freeze-thaw cycles, after 6 months at -20 °C and after the pasteurization process (differences to the initial concentration always lower than 10%). matrix effect ranged from 77.6% to 116.6%, recovery from 51.6% to 86.5%, and process efficiency from 46.8% to 79.0%. This LC-MS-MS assay was applied to screen samples from the largest Spanish milk bank and samples coming from drug addicted mothers. The developed method provided adequate sensitivity and performance characteristics to prove the presence of only caffeine in a small percentage of samples from milk donating nursing mothers and the presence or absence of most commonly used illicit drugs in breast milk from addicted lactating mothers.

Liquid chromatography-tandem mass spectrometry for fatty acid ethyl esters in meconium : Assessment of prenatal exposure to alcohol in two European cohorts

Fatty acid ethyl esters (FAEEs) in meconium emerged as a reliable, direct biological marker for establishing fetal exposure to ethanol. We developed an LC-MS/MS method for ethyl laurate, ethyl myristate, ethyl palmitate, ethyl palmitoleate, ethyl stearate, ethyl oleate, ethyl linoleate, ethyl linolenate, and ethyl arachidonate using ethyl heptadecanoate as the internal standard. The analytes were extracted from meconium with hexane, followed by solid-phase extraction with aminopropyl-silica columns. Chromatography was performed on a C(8) reversed-phase column using water/isopropanol/acetonitrile (20:40:40, v/v/v) as a mobile phase. A triple quadrupole mass spectrometer that monitored the transitions in multiple reaction-monitoring mode was used for the detection of the analytes. Limits of quantification (LOQs) varied between 0.12 and 0.20 nmol/g. Calibration curves were linear from LOQs to 50 nmol/g for all analytes, with a minimum r(2)>0.99. At three concentrations spanning the linear dynamic range, mean recoveries ranged between 53.6 and 86.7% for the different analytes. The validated method was applied to analysis of meconium in newborns of two European cities. The two cohorts presented with different prevalence of gestational ethanol consumption during pregnancy.

Biological matrices for the evaluation of exposure to environmental tobacco smoke during prenatal life and childhood

The measurement of nicotine and its major metabolites cotinine and trans-3´-hydroxicotinine together with other minor metabolites (e.g., cotinine N-oxide, cotinine, and trans-3´-hydroxicotinine glucuronides) in conventional and nonconventional biological matrices has been used as a biomarker to assess the exposure to environmental tobacco smoke during childhood. The determination of these substances in matrices such as amniotic fluid, meconium, and fetal hair accounts for prenatal exposure to cigarette smoking at different stages of pregnancy. Nicotine and its metabolites in cord blood, neonatal urine, and breast milk are useful for determining acute exposure to drugs of abuse in the period immediately before and after delivery. Cotinine measurement in children’s blood and urine and nicotine and cotinine measurements in children’s hair constitute objective indexes of acute and chronic exposure during infancy, respectively. However, for monitoring and categorizing cumulative exposure to environmental tobacco smoke during the entire childhood, including the prenatal period, the assessment of nicotine in teeth has been proposed as a promising noninvasive tool. This article reviews the usefulness of measurement of nicotine and its metabolites in different fetal and pediatric biological matrices in light of noninvasive collection, time window of exposure detection, and finally clinical application in pediatrics.

Assessment of chronic exposure to cigarette smoke and its change during pregnancy by segmental analysis of maternal hair nicotine

This study aimed to investigate the association between biomarkers of fetal exposure to smoking during the whole pregnancy, nicotine in maternal and newborns hair samples, and quantitative measurement of smoking intake and exposure evaluated by maternal self-reported questionnaire. Study subjects were 150 mothers and their newborns from a hospital in Barcelona. A questionnaire including smoking habits was completed in the third trimester of pregnancy and on the day of delivery. Nicotine content was measured in two subsequent segments of maternal hair accounting for the first and last months of pregnancy, and in fetal hair. The geometric mean of nicotine concentration in maternal hair discriminated between nonexposition (3.84 and 2.80 ng/mg in distal and proximal hair segment, respectively) and exposition to cigarette smoke during pregnancy (6.06 and 4.30 ng/mg in distal and proximal hair segment, respectively) (P<0.05), and between these two classes and active smoking (14.40 and 11.08 ng/mg in distal and proximal hair segment, respectively). Maternal hair nicotine was able to differentiate levels of exposure to tobacco smoke and levels of intake. Nicotine concentration in hair from newborns did not differentiate between exposure and nonexposure to environmental tobacco smoke (ETS) in nonsmoking mothers. Finally, chronic exposure to cigarette smoke during pregnancy, assessed by maternal hair nicotine, correlated negatively with anthropometric parameters of newborns.

Gas chromatography–mass spectrometry assay for the simultaneous quantification of drugs of abuse in human placenta at 12th week of gestation

We describe the development and validation of a method for the quantification of drugs of abuse, using gas chromatography-mass spectrometry (GC/MS), in human placenta. Concentration ranges covered were 5-500 ng/g for amphetamine, methamphetamine, MDMA, methadone, cocaine, benzoylecgonine, cocaethylene, morphine, 11-nor-9-carboxy-delta-9-tetrahydrocannabinol, nicotine, and cotinine. Intra-assay and inter-assay imprecisions were less than 15.7% for lower quality control samples and less than 14.9% for medium and high quality control samples. Recovery range was 36.2-83.7%. Placenta samples were kept at -80 degrees C until analysis; analytes were stable after three freeze-thaw cycles (samples stored at -20 degrees C). This accurate and precise assay has sufficient sensitivity and specificity for the analysis of specimens collected from women who voluntarily terminated their pregnancy at 12th week of gestation. The method has proven to be robust and accurate for the quantification of the principal recreational drugs of abuse in this period of the prenatal life. This is the first report that highlights the presence of drugs of abuse during the first trimester of gestation.

A comprehensive review of assay methods to determine drugs in breast milk and the safety of breastfeeding when taking drugs

AbstractMost of the licit and illicit drugs consumed by the breastfeeding woman pass into the milk and can modify the production, volume and composition of the milk, as well as hypothetically have short- and long-term harmful effects on the infant. There is much confusion in the scientific community regarding this issue: should a woman breastfeed her baby while continuing to use prescription drugs and/or drugs of abuse? There are many case reports of clinically significant toxicity in breast-fed infants from some substances used by mothers (such as irritability, vomiting, sedation, respiratory depression, shock), but there are too few data on studies conducted in breastfeeding women and their infants to make a realistic risk assessment. The objective measurement of a drug and/or metabolites in maternal milk is the first step when investigating the amount of drug exctreted in milk and subsequently calculating the daily dose administered to the breast-fed infant. The present review reports the analytical methods developed to detect different drugs in the breast milk, listing the principal characteristics and validation parameters, advantages and disadvantges. Furthermore, the mechanisms of drug transfer into breast milk are discussed, the correlation between the concentration of the drug in breast milk and potential adverse outcomes on the infant are described for each drug, and suggested harm minimization strategies and approved breastfeeding recommendations are indicated. Figure 

Neonatal Urinary Cotinine Correlates With Behavioral Alterations in Newborns Prenatally Exposed to Tobacco Smoke

Altered behavior due to prenatal smoke exposure was examined in 25 neonates born from smoking mothers who consumed at least 5 cigarettes/d during the entire gestation. Data were compared with 25 matched neonates born from nonsmoking mothers. Neonatal behavior was evaluated using the Brazelton Neonatal Behavioral Assessment Scale (BNBAS). Antenatal exposure to tobacco smoke at the end of the pregnancy was determined by measurement of urinary cotinine. Newborns from smoking mothers showed significant lower scores in various BNBAS items compared with neonates from nonsmoking mothers. A strong correlation was observed between infant irritability and urinary cotinine in newborns from smoker and nonsmoking mothers and with number of daily smoked cigarettes and maternal nicotine daily intake of infants exposed to active maternal smoking. Linear regression analysis showed that urinary cotinine was the best predictor of infant irritability (r2 = 0.727). The latter was also associated to the neonates low level of attention and poor response to inanimate auditory stimuli. Among infants from nonsmoking mothers, paternal smoking significantly correlated with infant urinary cotinine and infant irritability, being also the best predictor of irritability (r2 = 0.364). Neonatal behavior can be significantly altered in a dose-dependent manner even after modest prenatal exposure to tobacco smoke.