Using time-of-flight secondary ion mass spectrometry (ToF-SIMS) and matrix assisted laser desorption/ionization mass spectrometry (MALDI-MS) for investigations on single hair samples to solve the contamination versus incorporation issue of hair analysis in the case of cocaine and methadone.

Drug testing in hair is a controversial subject of discussion. Claims that decontamination protocols could generate false-positive samples, by washing contamination in hair, have unsettled many toxicologists. At least for zolpidem (known for showing only minor contamination), it could be shown that differentiation of the drug incorporated via the bloodstream from contamination was possible. The current work addresses cocaine and methadone, known for their high concentrations and contamination issues. Longitudinally and cross-sectioned samples of drug-soaked hair, consumer hair and cocaine powder contaminated hair were investigated using time of flight-secondary ion mass spectrometry (ToF-SIMS) and matrix-assisted laser desorption/ionization-mass spectrometry (MALDI-MS). In addition, the resulting wash solutions were investigated using LC-MS/MS. Differentiation of contamination from incorporation was possible for soaked and consumer hair samples. Therefore, contamination could be localized in the superficial compartments of hair and could be removed using strong wash protocols. In the case of powder contaminated hair samples, a small amount of cocaine remained in the inner structures even after the application of the strongest wash protocols. However, taking into consideration the differences in their behavior during decontamination steps compared to both soaked and authentic hair samples, the validity of this contamination protocol (rubbing cocaine powder into hair) must be questioned. Furthermore, when using cut-off values and metabolite ratios (from routine hair analysis), the differentiation of incorporation from contamination was possible also for all our experimental samples in this study. Inclusion of metabolites and application of cut-off values are therefore a must in routine hair analysis.

Hair Analysis: Contamination versus Incorporation from the Circulatory System-Investigations on Single Hair Samples Using Time-of-Flight Secondary Ion Mass Spectrometry and Matrix-Assisted Laser Desorption/Ionization Mass Spectrometry.

Contamination is a highly controversial issue in hair analysis. Therefore, hair testing protocols typically include wash steps to remove contamination. However, recent studies claim that washing could also lead to permanent incorporation of contaminants into hair, thus questioning the validity of hair testing at all. In the present study, matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) with longitudinal sectioning of single hairs and different decontamination protocols was used to reveal differences between the incorporation of a substance into hair from external sources and an incorporation via bloodstream. Single hairs were longitudinally sectioned using a custom-made sample holder. Data were acquired with MALDI-MS by rastering each hair individually. Single hair samples from drug users, blank hairs, and zolpidem- and zolpidem-D6-soaked hairs were investigated. Different published washing protocols were tested, and an in-house washing protocol was developed. For images with higher spatial resolution, time-of-flight secondary ion mass spectrometry (ToF-SIMS) was used. Longitudinal sectioning of hairs dramatically increased sensitivity; even single-dose administrations of zolpidem in single hairs could thus be detected using MALDI-MS. Zolpidem from external sources could be detected in large quantities in superficial hair structures. Zolpidem from consumer hairs, proposed to be strongly bound to inner hair structures, could not be completely removed even by the strongest tested decontamination protocol, whereas zolpidem-soaked hairs could be cleared almost completely with the developed in-house wash protocol. The applied methods allowed a first insight into the connection of decontamination protocols and wash-in phenomena in hair analysis. Further studies with other drugs are necessary to assess the general validity of these findings.

Insights into the Decontamination of Cocaine-Positive Hair Samples.

A highly discussed step in hair sample preparation for forensic analytics is the applied decontamination. The here presented investigations aim to gain insight and give recommendations on how to conduct this decontamination for the analysis of cocaine consumption in hair. Key insights were gained from the investigation of cocaine consumer hair, which was artificially contaminated in a humid atmosphere with 13C6 labelled cocaine and from cocaine powder contaminated hair. Several decontamination protocols were investigated, whereby the usage of a decontamination protocol consisting of multiple short repetitive washes allowed to visualize the wash out of (13C6-) cocaine. Multiple methanol washes proved to be an efficient and simple decontamination approach. Our findings showed that decontamination protocols can successfully wash out recent cocaine contaminations. They were observed to be rather quickly washed out, whereas cocaine from consumption or "older" cocaine contaminations were shown to eliminate both at a constant rate (from inner hair compartments). Thus, the usage of decontamination protocols to differentiate between consumption and contamination was shown to be limited. As contamination can happen any time at any level, only the application of elaborated decision trees, based on cocaine metabolite ratios and thresholds, can provide the distinction between consumption and contamination. Thus, the authors highly recommend the usage of such tools on all hair samples analyzed for cocaine consumption.