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

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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.

Key Words: heroin, diacetylmorphine, 6-monoacetylmorphine, meconin, umbilical cord tissue

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Introduction

From 1992 to 2010, the proportion of admissions into substance abuse treatment facilities because of prescription opiate addiction has risen from 1% to 8.6% of total admissions, whereas the proportion of cocaine and methamphetamine-based admissions have dropped or remained steady. The Center for Substance Abuse Research further noted that during the same time period there were statistically significant increases of self-reported chronic nonmedical use of prescription pain relievers. In response to those trends, measures to contain the prescription opiate epidemic have been implemented but have led to the unintentional consequence of diverting this opiate-addicted population to a low cost and readily available supply of heroin. One study demonstrated that since the introduction of extended-release oxycodone (an abuse-deterrent formulation strategy embraced by the FDA), the abuse of oxycodone decreased but was unfortunately accompanied with a 42% increase of heroin use in the study population. Included in this dangerous trend is an increase of opiate exposed newborns that exhibit a wide array of negative signs and symptoms called Neonatal Abstinence Syndrome (NAS).

NAS, although implicated from the abuse of other drugs, is typically associated with the sudden neonatal withdrawal from opiates. Symptoms include irritability, hypertonia, tremors, feeding intolerance, emesis, watery stools, seizures, and respiratory distress (Hudak et al). Between the years 2000 and 2009, the incidence of NAS has risen from 1.2 to 3.4 per 1000 births. Patrick et al noted that the average length of stay for all births during the study period was approximately 3 days, whereas the newborns identified with NAS had an average length of stay of 16 days creating added burden on hospital resources that are disproportionally covered by State programs.

Screening newborns for drugs of abuse has historically been accomplished using maternal self-report and analysis of neonatal biological specimens such as urine, meconium, and umbilical cord. Maternal self-report may be unreliable because of deception and the stigma of illicit drug use and urine testing has a limited detection window of only a couple of days. Meconium, the first fecal matter produced by the newborn, has been used for the past 20 years for drugs of abuse testing because of its relative availability (78%–90%) and its long detection window (up to 20 weeks). Umbilical cord testing has rapidly replaced meconium over the past 5 years as the newborn toxicology gold standard because of its universal availability (every baby has sufficient umbilical cord for testing), shortened turnaround time (umbilical cord is collected immediately after birth while meconium may take several days to pass), and improved chain of custody integrity (meconium requires multiple collections by multiple collectors over multiple shifts, whereas umbilical cord is a single-step collection).

The first report of using umbilical cord segments to screen newborns for opiates exposure was given by Montgomery et al, where they reported finding opiates in matched pairs of meconium and umbilical cord with a 95% agreement between the 2 specimen types. They used immunoassay methods (ELISA), and these results were verified using a modified meconium gas chromatography–mass spectrometric method. A large trial of 498 umbilical cords was used to survey 2 populations (Newark, NJ and Salt Lake City, UT), and these data were used to evaluate the sensitivity (91%) and specificity (98%) of the immunoassay compared with the modified gas chromatography–mass spectrometric method. The following year, a more sensitive liquid chromatography–tandem mass spectrometry method was published by the Chemistry and Metabolism group at the National Institute on Drug Abuse with a reported limit of detection (LOD) of 1.0 ng/g for 6-monoacetylmorphine (6MAM) and 2.5 ng/g for codeine and morphine but they did not report finding any unique heroin metabolites in authentic umbilical cord specimens. Our laboratory used a slightly modified version of this method until October 2012, and feedback from the field suggested that improvements in sensitivity were needed for identifying heroin-exposed newborns.

Heroin (diacetylmorphine) is rapidly metabolized to 6MAM which is then quickly hydrolyzed to morphine. In urine, the detection window for 6MAM is less than 1 day with morphine persisting for several days afterward. The presence of 6MAM is unique to heroin but morphine may be present because of the ingestion of morphine or codeine. Because of the rapid elimination of 6MAM, routinely only morphine is detected. Additionally, in some instances, mothers are administered morphine during labor and delivery, which confounds interpretations in the absence of a unique heroin metabolite.

A similar situation exists in several European countries where pharmaceutical grade diacetylmorphine is commonly used. In that environment, 6MAM is not unique for illicit heroin use. Morley et al demonstrated that the detection of meconin, a metabolite of the illicit heroin contaminant noscapine, was an effective strategy to discriminate illicit heroin use from pharmaceutical grade diacetylmorphine use. Unexpectedly, the detection of meconin outperformed the detection of 6MAM by 69% in a survey of 300 urine specimens from known illicit heroin users. The findings of Morley et al prompted the question, would the inclusion of meconin in the umbilical cord assay improve the identification of in utero heroin exposure?

We propose that improvements of the analytical sensitivity and the inclusion of meconin in the LC-MS/MS opiates assay will augment our efforts to identify heroin-exposed newborns. We will present a fully validated LC-MS/MS method for the analysis of codeine, morphine, 6MAM, and meconin in umbilical cord and report here for the first time the presence of unique heroin metabolites in authentic umbilical cord specimens. We will analyze a selection of authentic umbilical cord specimens to compare this method to the previous method. Finally, we will summarize our findings for the specimens received by our laboratory for 1 year using the new method.