Interpretation of hair findings in children after methadone poisoning

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Abstract

Methadone is not licensed for use in children though it can be employed for the management of neonatal opiate withdrawal syndrome. During the last 2 years, our laboratory has been asked to test for methadone and EDDP, its major metabolite, in hair from children that were admitted to hospital unconscious and where methadone had already been identified in a body fluid (4 cases) or where the children were deceased and evidence of methadone overdosage having already been established (2 cases). In all of these cases, segmental analysis revealed approximately the same amount of drug along the hair lock. As a consequence, contamination was considered as an issue and interpretation of the results was a challenge that deserves particular attention.

After decontamination with dichloromethane and segmentation the hair was cut into small pieces, incubated overnight at 40 °C, liquid–liquid extracted and analysed with LC–MS/MS, using 2 transitions per compound. The LOQ for both methadone and EDDP was 10 pg/mg.

In the first series involving children admitted to hospital, the following results were obtained:

  • case 1: 4 × 1 cm section, methadone at 0.05–0.08 ng/mg, no EDDP detected,

  • case 2: 4 × 1 cm section, methadone at 0.13–0.15 ng/mg, EDDP at 0.02 ng/mg,

  • case 3: 3 × 1.5 cm section, methadone at 0.07–0.09 ng/mg, EDDP at 0.01–0.03 ng/mg,

  • case 4: 6 × 2 cm section, methadone at 0.06–0.13 ng/mg, EDDP at 0.02–0.03 ng/mg.

The following concentrations were obtained from the children who had died following a methadone overdose:

  • case 5: 2 × 2 cm section, methadone at 0.53–0.58 ng/mg, no EDDP detected,

  • case 6: 4 × 1 cm section, methadone at 0.44–0.77 ng/mg, EDDP at 0.04–0.06 ng/mg.

The first observation is that all these concentrations are low by comparison with those observed in adults on methadone maintenance therapy. However, the more surprising observation is the relative homogenous concentrations along the hair locks in each specific case. This raises concerns around the possibility that contamination could have occurred prior to sampling and makes it hard to reach a conclusion regarding the possibility of repeated methadone exposure in the months prior the incidents.

In these cases it was impossible to conclude that the children were deliberately administered methadone. The results of the analysis of hair could indicate that they were in an environment where methadone was being used and where the drug was not being handled and stored with appropriate care. The homogenous concentrations found on segmental analyses could be indicative of external contamination that may have arisen not only from direct contamination with the drug but also via contamination with body fluids at the post mortem or from sweat produced close to the time of the incident. In view of these results we concluded that a single determination should not be used firmly to discriminate long-term exposure to a drug.

Introduction

The major advantage of hair testing compared to urine or blood testing for drugs is that it has a larger surveillance window (weeks to months, depending on the length of the hair shaft compared to 2–4 days for most drugs in blood and urine). For practical purposes, the two tests complement each other. Urinalysis and blood analysis provide short-term information of an individual's drug use, whereas long-term histories are accessible through hair analysis.

By providing information on exposure to drugs over time, hair analysis may be useful in verifying self-reported histories of drug use in any situation in which a history of past rather than recent drug use is desired. In addition, hair analysis may be especially useful when a history of drug use is difficult or impossible to obtain.

Numerous forensic applications have been described in the scientific literature where hair analysis was used to document historic drug use or exposure: suspicious death, evidence of drug administration, evidence of long-term poisoning, discrimination between single and chronic exposure, demonstration of tolerance, pattern of drug use, crimes committed under the influence of drug [1].

Although it is generally agreed that the qualitative results from hair analysis are valid, the interpretation of the results is still under debate owing to unresolved questions such as the influence of external contamination. More research is required before all of the scientific questions associated with hair drug testing will be satisfied. There remains a lack of consensus among those scientists active in the field of hair testing on how to exclude external contamination.

Contamination of hair would be a major problem in a negative specimen where the apparent detection of a drug and/or metabolites(s) would lead to a positive interpretation. However, it is unlikely that anyone would intentionally or accidentally apply anything to his or her hair that would contain a drug. Thus, in reality, the most crucial issue in the interpretation of hair analysis is the avoidance of technical and evidentiary false-positives. Technical false-positives are those caused by contamination occurring during the collection, processing and analysis of specimens, whilst evidentiary false-positives are caused by passive exposure to the drug. Approaches for preventing evidentiary false-positives due to external contamination of the hair specimens have been described since 1992 [2]. These criteria do not endorse a general acceptance [3], [4]. Excluding laboratory mistakes, a false positive hair result can be observed in case of contamination from environmental pollution or after drug incorporation into the hair from the individual body fluids, such as sweat or putrefactive fluid.

Most laboratories use a wash step; however, there is no consensus or uniformity in the washing procedures. Among the agents used in washing are detergents such as shampoo, surgical scrubbing solutions, surfactants such as 0.1% sodium dodecylsulfate, phosphate buffer or organic solvents such as acetone, diethyl ether, methanol, ethanol, dichloromethane, hexane or pentane of various volumes for various contact times. From the papers in the scientific literature, a single washing step is used; although a second identical wash is sometimes performed. If external contamination is found by analysing the wash solution, the washout kinetics of repeated washing can demonstrate that contamination is rapidly removed. Baumgartner and Hill [2], published that the concentration of drug in the hair after washing should exceed the concentration in the last wash by at least 10 times. This was recently confirmed by Tsanaclis and Wicks [5]. It has also been proposed that hair should be washed three times with phosphate prior to analysis to remove any possible external contamination and that the total concentration of any drug present in the three phosphate washes should be greater than 3.9 times the concentration in the last wash [2]. Obviously, washing removes drug from the interior as well as from the exterior surface of hair during decontamination procedure [2].

According to Romano et al. [6], even using the most sophisticated decontamination procedures, it is not possible to distinguish a drug-contaminated subject from an active user. However, these results were challenged by Cairns et al. [7]. Thus, while a negative result excludes both chronic use and contact with drugs, a positive result cannot be interpreted as a sure sign of drug repetitive exposure.

Detection of drug metabolite(s) in hair, whose presence could not be explained by hydrolysis or environmental exposure, was proposed to unequivocally establish that internal drug exposure had occurred. Cocaethylene and nor-cocaine would appear to meet these criteria, as these metabolites are formed when cocaine is metabolised [8]. Because these metabolites are seldom found in illicit cocaine samples, they would not be present in hair as a result of environmental contamination, and thus their presence in hair may be considered as a marker of cocaine exposure. This procedure can be extended to other drugs, such as THC-COOH for cannabis [9]. However, until now, specific metabolites for numerous drugs (opiates, amphetamines, GHB, benzodiazepines …) have not been identified in hair.

As it can be observed from the scientific literature [2], [3], [4], [5], [6], [7], [8], several approaches have been proposed to get round the contamination scenario. However, all of these approaches are dealing with living subjects and none have been developed for post mortem specimens, collected under poorly controlled conditions. These references consider environmental contamination but not to incorporation via contact with body fluids, where metabolites can be present as a result of their presence in the fluids that contaminated the hair.

The authors present here 6 cases of methadone poisoning, where artefacts could not be excluded and the corresponding interpretative issues, which represent a first proposal to avoid false conclusions.

Section snippets

Case 1

The boy was taken to hospital unconscious where a urine sample was obtained and confirmed the presence of methadone. The police requested testing of a hair sample for any evidence of previous methadone administration. A strand of hair that was 4 cm in length and blonde in colour was collected.

Case 2

Parents were suspected of administering methadone to a 16 months old child. A strand of hair that was 5 cm in length and blonde in colour was collected.

Case 3

This case involved a suggestion that methadone was

Chemicals and reagents

Acetonitrile, isopropanol, n-heptane, and methylene chloride were HPLC grade (Merck, Darmstadt, Germany). Chemicals for the ammonium formate used in the mobile phase, ammonium chloride buffer used for the extraction – (NH4)2Cl, adjusted to pH 9.5 – and formic acid were purchased from Fluka (Saint-Quentin Fallavier, France). Methadone-d3 and EDDP-d3 were purchased from Promochem (Molsheim, France).

Extraction

Hair strands were decontaminated twice using methylene chloride (5 ml, 2 min) and then segmented.

Results and discussion

To be considered positive, the specimens had to produce transition ratios within 20% of those of the calibration standards.

Linearity was observed for methadone concentrations ranging from 0.05 to 10 ng/mg with a correlation coefficient of 0.9988. Within-batch precision at 1 ng/mg was 11.8% and the relative extraction efficiency was 76%. The limit of detection was 2 pg/mg, with a limit of quantitation of 10 pg/mg. Linearity was observed for EDDP concentrations ranging from 0.01 to 1 ng/mg with a

Conclusion

The findings reported here emphasize that with a single hair result it is impossible to determine the amount of drug that was used during periods removed in time from the time of sampling. To enable more reliable assessment of hair findings in determining if there is evidence of prior use or administration then multi-sectional analyses should be encouraged, with homogenous results being considered indicative of contamination. In some cases, hair analysis can be equivocal, and results should be

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