Joseph Jones

Prevalence of Fatty Acid Ethyl Esters in Meconium Specimens

BACKGROUND Fetal alcohol syndrome (FAS), alcohol-related birth defects (ARBDs), and alcohol-related neurodevelopment disorders (ARNDs) in neonates are often the result of maternal alcohol consumption during pregnancy. Facial characteristics are associated with FAS, but ARBDs and ARNDs are more difficult to diagnose. Fetal exposure to alcohol can cause central nervous system dysfunction, pre- and postnatal growth problems, cardiac defects in neonates, and attention deficit disorders and mental retardation in older children. To date, diagnosis of fetal alcohol effect has depended largely on maternal interview, although clinical tests are becoming more widely used. Fatty acid ethyl esters (FAEEs) are formed in the body by esterification of ethanol with free fatty acids and trans-esterification of glycerides and have been detected in the meconium of newborns. This report estimates the prevalence of fetal alcohol exposure in two populations by detecting FAEEs in meconium. METHODS We analyzed the prevalence of FAEEs in the meconium of two separate groups of neonates by use of solid-phase extraction and analysis by gas chromatography-mass spectrometry in the chemical ionization mode. In the first study, meconium samples were taken anonymously from babies born in a large, regional perinatal center in Hawaii. In the second study, specimens were obtained from infants admitted to six different newborn intensive care units within the state of Utah. RESULTS In the first study, 73 of 436 (16.7%) meconium specimens tested were considered positive for FAEEs. When broken down into quartiles, the mean total FAEEs measured were 1,059, 3,133, 6,628, and 62,115 ng/g. In the second study, 35 of 289 (12.1%) specimens were considered positive. When broken into quartiles, the mean total FAEEs were 1,139, 3,067, 7,674, and 50,143 ng/g. The overall FAEE profiles of the two study sets were remarkably similar. CONCLUSION In an adequate meconium specimen, a total FAEE concentration >10,000 ng/g may indicate that the newborn has been exposed to significant amounts of alcohol during pregnancy.

The detection of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanol in human dried blood spots

Phosphatidylethanol, a series of abnormal phospholipids formed in the presence of ethanol and phospholipase D, has gained popularity as a long-term biomarker of ethanol ingestion. A liquid chromatography tandem mass spectrometric method for a specific, prevalent isomer, 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanol, was developed and validated using dried blood spots. Dried blood spots offer numerous advantages over venipuncture including reduced costs, invasiveness and discomfort. Dried blood spots were prepared from authentic whole blood specimens that had been tested using a previously published procedure. Comparison of the results from the two assays demonstrated excellent correlation. The data suggest that dried blood spots may be a useful tool for the detection of alcohol abuse and abstinence monitoring.

Ethyl glucuronide in hair and fingernails as a long-term alcohol biomarker

Aims This study aimed to evaluate the performance of ethyl glucuronide (EtG) in hair and fingernails as a long-term alcohol biomarker. Design Cross-sectional survey with probability sampling. Setting Midwestern United States. Participants Participants were 606 undergraduate college students between the ages of 18 and 25 years at the time of selection for potential study participation. Measurements EtG concentrations in hair and fingernails were measured by liquid chromatography-tandem mass spectrometry at three thresholds [30 picograms (pg) per milligram (mg); 20 pg/mg; and 8 pg/mg]. Any weekly alcohol use, increasing-risk drinking and high-risk drinking on average during the past 12 weeks was assessed by participant interview using the time-line follow-back method. Findings In both hair and fingernails at all three EtG thresholds, sensitivity was greatest for the high-risk drinking group [hair: 0.43, confidence interval (CI) = 0.17, 0.69 at 30 pg/mg, 0.71, CI = 0.47, 0.95 at 20 pg/mg; 0.93, CI = 0.79, 1.00 at 8 pg/mg; fingernails: 1.00, CI = 1.00–1.00 at 30, 20 and 8 pg/mg] and specificity was greatest for any alcohol use (hair: 1.00, CI = 1.00, 1.00 at 30 and 20 pg/mg; 0.97, CI = 0.92–0.99 at 8 pg/mg; fingernails: 1.00, CI = 1.00–1.00 at 30, 20 and 8 pg/mg). Areas under the receiver operating characteristic curves were significantly higher for EtG concentration in fingernails than hair for any weekly alcohol use (P = 0.02, DeLong test, two-tailed) and increasing-risk drinking (P = 0.02, DeLong test, two-tailed). Conclusions Ethyl glucuronide, especially in fingernails, may have potential as a quantitative indicator of alcohol use.

Determination of amphetamine and methamphetamine in umbilical cord using liquid chromatography-tandem mass spectrometry

The use of meconium as a drug-screening matrix for newborns has been the gold standard of care for the past two decades. A recent study using matched pairs of meconium and umbilical cord demonstrated a high degree of agreement. The use of liquid chromatography-tandem mass spectrometry as a means to confirm amphetamines presumptive positive umbilical cord specimens for amphetamine and methamphetamine is described here for the first time. The limit of detection for both compounds was 0.2 ng/g. The limit of quantitation for both compounds was 0.6 ng/g. The assay was linear for both compounds up to 100 ng/g.

Detection of Drugs in Nails: Three Year Experience

Nails (fingernails and toenails) are made of keratin. As the nail grows, substances incorporate into the keratin fibers where they can be detected 3-6 months after use. Samples are collected by clipping of 2-3 mm of nail from all fingers (100 mg). We present drug testing results from 10,349 nail samples collected from high-risk cases during a 3-year period of time. Samples were analyzed by validated analytical methods. The initial testing was performed mostly using enzyme-linked immunosorbent assay, but by liquid chromatography-tandem mass spectrometry (LC-MS-MS) as well. Presumptive positive samples were subjected to confirmatory testing with sample preparation procedures including washing, pulverizing, digestion and extraction optimized for each drug class. The total of 7,799 samples was analyzed for amphetamines. The concentrations ranged from 40 to 572,865 pg/mg (median, 100-3,687) for all amphetamine analytes. Amphetamine and methamphetamine were present in 14% of the samples, 22 samples were positive for 3,4-methylenedioxymethamphetamine (0.3%), 7 for methylenedioxyamphetamine (0.09%) and 4 for 3,4-methylenedioxy-N-ethylamphetamine (0.05%). Cocaine and related analytes were found in 5% samples (7,787 total), and the concentration range was 20-265,063 pg/mg (median 84-1,768). Opioids overall ranged from 40 to 118,229 pg/mg (median 123-830). The most prevalent opioid was oxycodone (15.1%) and hydrocodone (11.4%) compared with 1.0-3.6% for the others, including morphine, codeine, hydromorphone, methadone, 2-ethylidene-1,5-dimethyl-3,3-diphenylpyrrolidine and oxymorphone. Carboxy-Δ-9-tetrahydrocannabinol positivity rate was 18.1% (0.04-262 pg/mg, median 6.41). Out of 3,039 samples, 756 were positive (24.9%) for ethyl glucuronide (20-3,754 pg/mg, median 88). Other drugs found in nails included barbiturates, benzodiazepines, ketamine, meperidine, tramadol, zolpidem, propoxyphene, naltrexone and buprenorphine. Nail analyses have become a reliable way of determining the long-term use and abuse of drugs. Extraction techniques are simple and produce accurate and precise results. Sensitive analytical instrumentation, mainly LC-MS-MS, allows for detection of femtogram (10(-15) g) quantities of substances in nails. Samples were from a high-risk population, therefore the extraordinary positivity rate was observed.

Determination of methamphetamine enantiomer composition in human hair by non-chiral liquid chromatography–tandem mass spectrometry method

Chiral separation is crucial for investigating methamphetamine positive cases. While (S)-(+)-enantiomer of methamphetamine (S-MAMP) is a schedule II controlled substance, (R)-(-)-enantiomer (R-MAMP) is an active ingredient of a few over-the-counter drugs in the United States. Among biological specimen types, hair provides greater detection window than blood, urine or oral fluid, and are therefore regarded with particular interest. Herein we describe a novel non-chiral liquid chromatography-tandem mass spectrometry (LC-MS/MS) method to directly determine methamphetamine enantiomeric composition (percentage) in hair specimens. Hair samples were washed once with acetone, powdered, incubated overnight at 53°C in 0.1M hydrochloric acid (HCl), and subjected to a solid phase extraction (SPE). The extracts were derivatized using Marfeys reagent at 53°C for 60min. The final mixture was analyzed by LC-MS/MS. Chromatographic separation was achieved using a C18 Kinetex analytical column and 60% (v/v) aqueous methanol as mobile phase (isocratic). Triple quadrupole mass spectrometer was equipped with an electro-spray ionization (ESI) source operating in negative mode and the chromatograms were acquired using a multiple-reaction monitoring (MRM) approach. The results were expressed as ratio of R- to S-MAMP and then derived to composition percentages without requiring quantitating each enantiomer. The method was precise and accurate across 0-100% S-composition at a range of 80-18,000pg/mg. The performance of the new method was compared with an (S)-(-)-N-trifluoroacetylprolyl chloride (S-TPC) derivatization and gas chromatography-mass spectrometry (GC-MS) method on authentic methamphetamine-positive hair samples. Not only the new Marfeys reagent approach presented satisfactory correlation with the S-TPC approach, but it also exhibited significantly improved quality (e.g., S/N) of the chromatograms. In summary, our protocol employs cost effective and minimally hazardous Marfeys reagent to derivatize trace amounts of methamphetamine extracted from hair samples and a non-chiral LC-MS/MS approach to separate and identify the two enantiomers. The method allows determination of the methamphetamine enantiomeric composition without requiring quantitation of each enantiomer and is therefore well suited for further investigate previously determined methamphetamine positive cases. This method represents a viable tool for evaluation of long-term drug exposure.

Detection of Codeine, Morphine, 6-Monoacetylmorphine, and Meconin in Human Umbilical Cord Tissue: Method Validation and Evidence of In Utero Heroin Exposure

Background: Heroin abuse is a significant public health issue and is on the rise because of the unintended consequences of strengthening controls for nonmedical use of prescription pain killers. Included in this trend is an increase in opiate exposed newborns that are particularly vulnerable to a number of negative health outcomes. Methods: After presenting a fully validated liquid chromatography–tandem mass spectrometric method for codeine, morphine, 6-monoacetylmorphine, and meconin, a metabolite of the heroin contaminant noscapine, we compared the outcome of 46 authentic umbilical specimens with the results generated using a previous less sensitive method that did not include meconin. Additionally, we provided a summary of opiate finding from a year-long survey of specimens received into a commercial reference laboratory. Results: The limits of detection for all 4 compounds were 0.1 ng/g, the limit of quantitation was 0.2 ng/g, and the assay was linear from 0.2 to 10.0 ng/g. Of the 46 comparative specimens, this method improved the identification of heroin exposure from 2 to 5, and the year-long survey identified 86 heroin-exposed newborns with 11 of them identified by the sole identification of meconin. Conclusions: This study demonstrated that a more sensitive analytical platform and the inclusion of meconin in the opiates assay improved the ability to distinguish between in utero heroin exposure and maternal administration of codeine or morphine.

The detection of caffeine and cotinine in umbilical cord tissue using liquid chromatography–tandem mass spectrometry

A liquid chromatography-tandem mass spectrometry method for the simultaneous detection of cotinine and caffeine in umbilical cord was fully validated. The analytes were quantified using multiple reaction monitoring and corresponding stable isotope internal standards (cotinine-d3 and caffeine-13C3). The method demonstrated acceptable imprecision (<12%) and bias (<13%). The limit of detection was 4 ng g−1 and 40 ng g−1 for cotinine and caffeine, respectively. The determined relative matrix effects for cotinine and caffeine were 3.0% and 3.2%, respectively. The method was used to successfully analyze two authentic umbilical cords that were matched with corresponding meconium specimens that had been analyzed for cotinine and caffeine using a previously published method. A simple, one-step collection procedure and the availability of specimen for every donor make umbilical cord a simple alternative for newborn toxicology.

Discordant Umbilical Cord Drug Testing Results in Monozygotic Twins

Our laboratory received segments of umbilical cord that originated from identical twins for routine toxicology analysis. The specimens were analyzed multiple times by liquid chromatography tandem mass spectrometry. The umbilical cord from newborn #1 was positive for hydromorphone only (1.06 ng/g), and the umbilical cord from newborn #2 was positive for hydromorphone (0.81 ng/g) and benzoylecgonine (5.41 ng/g). The hydromorphone results are consistent with maternal administration of hydromorphone; however, the cause of the discrepant benzoylecgonine results in the umbilical cords from the identical twins is unknown.

The utility of collateral student drinking reports: Evidence from a biomarker study.

INTRODUCTION Researchers have increasingly used collateral informants to validate the reports provided by primary research subjects. We assessed the utility of collateral informants for college students in a study that incorporates biomarkers to validate student reports of recent drinking behavior. METHODS Students from a Midwestern university were randomly selected for a study in which they provided 90-day Timeline Followback data, hair and fingernail specimens for ethylglucuronide (EtG) testing, and information about collateral (friends or peers) informants who were familiar with their drinking behavior. We compared summary measures of recent drinking to collateral informant reports for the subset of 72 students who were selected to participate in the collateral validation process who had complete measures. Kappa, weighted kappa, and McNemar tests were performed to evaluate levels of agreement. We compared levels of use indicated by each informant within the context of EtG findings. We also compared respondent and collateral reports with respect to heavy drinking directly to EtG test results. RESULTS There was considerable overlap between the reports provided by the student participants and their collateral informants. Within the context of EtG-informed analyses, collaterals rarely provided new information about heavy use beyond that provided by the study subjects. CONCLUSIONS Collateral informants have limited utility in non-clinical studies of heavy drinking in randomly selected college students.