Improved forensic hair evidence for drugs of abuse by mass spectrometry

Forensic hair analysis can be used as alternative evidence next to body fluids, and to obtain retrospective timeline information of an individual’s drug exposure. Chapter 1 describes the general concepts of drug incorporation into hair, external contamination, and the current status and limitations of hair analysis methods are introduced. Furthermore, an overview of ambient ionization techniques is given, with emphasis on direct analysis in real time (DART).
The instrumentation, ionization mechanisms, and application range of DART are presented. Scientific challenges and objectives to improve forensic hair evidence are formulated, which formed the basis of the research presented in this thesis.

A major issue in forensic hair analysis is the possibility of false-positive results due to external contamination. In Chapter 2, an evidence-based evaluation of decontamination protocols for the removal of cannabinoid contamination is presented, mainly focused on
Δ9-tetrahydrocannabinol (THC). Different solvents were extensively tested for their ability to remove cannabinoid contamination originating from cannabis smoke or indirect contact with cannabis plant material. After selection of the most efficient solvents, different sequential wash steps were tested on externally contaminated blank hair samples. Finally, application of the three best performing protocols on cannabis users’ hair, both as such and after deliberate contamination, resulted in removal of all contamination without removing incorporated THC. From the detailed scientific evidence reported in this chapter, a protocol using a single methanol wash followed by a single aqueous SDS solution is recommended to remove external cannabis contamination.

A novel approach for the analysis of intact locks of hair consisting of DART combined with high resolution mass spectrometry (HRMS) is developed in Chapter 3. DART–HRMS settings were optimized for the analysis of THC and the accuracy of the probed hair zone was investigated using spiked blank hair samples. Intact locks of hair could be longitudinally scanned without the need of extensive sample preparation, resulting in analysis times of only minutes. Detection of THC was achieved in several hair samples from cannabis users. A quantitative liquid chromatography (LC)–MS/MS method was developed, in-house validated, and used to confirm the presence of THC in drug user hair samples. With a retrospective timeline accuracy of ±2 weeks, a significant improvement over conventional segmented hair analysis was achieved. Moreover, differentiation between zones of different THC content within a DART hair scan could be made, indicating possibilities for retrospective assessment of time of drug use.

The DART hair scan method has been improved and expanded in Chapter 4. Targeted detection of four commonly used drugs of abuse (amphetamine, cocaine, MDMA and THC) with structural confirmation was achieved by data-dependent product ion scans. Simultaneously,
full-scan high-resolution data was obtained and retrospectively interrogated versus a list of more than a hundred, less common, drugs of abuse and occasionally abused pharmaceutical drugs. The hair scan method was validated for the analysis of cocaine against an accredited LC–MS/MS method and the detection limit for cocaine was found to comply with the cut-off value of 0.5 ng/mg. Hair samples of 10 different drug users were analyzed. Next to detection of the four targeted drugs of abuse, retrospective data interrogation revealed several additional hits. The detected substances correlated well with reported drug use and by the detection of several metabolites, drug use could be unambiguously proven. The retrospective timeline accuracy was further improved by use of a high spatial resolution DART exit cone, which yielded a DART spot size corresponding to approximately 10 days of hair growth.

When direct and/or ambient ionization techniques are used to analyze intact hair samples, endogenous isobaric ions can overlap with compounds of interest and yield
false-positive results. The selectivity of four MS instruments with different mass analyzers (orbitrap, quadrupole orbitrap, triple quadrupole, time-of-flight) was evaluated in Chapter 5 by DART analysis of THC from hair samples. To avoid overlap of THC with isobaric ions originating from the hair matrix, a mass resolution of at least 30,000 FWHM was necessary. The use of travelling wave ion mobility spectrometry (TWIMS) resulted in increased selectivity by separation of isobaric ions based on their drift times. A triple quadrupole instrument in multiple reaction monitoring (MRM) mode was found to have the best sensitivity, however, the used transitions were not specific enough for use on drug user hair samples. Thus the selectivity needed to indisputably differentiate THC from endogenous isobaric ions in drug user hair samples could only be achieved by the high resolution of the tested orbitrap MS instruments.

Chapter 6 demonstrates the application of forensic hair analysis techniques to veterinary control. Timeline information could be obtained from veterinary hair samples. For this purpose, a UPLC–MS/MS hair analysis method was adapted and optimized for smaller sample sizes.
After validation of the method, segmented hair samples obtained from clenbuterol-treated calves using the forensic hair sampling protocol were analyzed and clenbuterol concentration profiles along the hair samples could be obtained. Assessment of the average growth rate of calf tail hair enabled retrospective determination of time of clenbuterol administration.
The estimated time of administration was reproducible when analyzing sub-samples taken from the same lock of hair and duplicate locks of hair, and in good correlation with the actual treatment.

Through the research presented in this thesis, novel approaches in hair analysis have been developed and the value of forensic hair evidence improved considerably. In Chapter 7, the main achievements of this thesis are discussed in detail and an insight in the future perspectives of hair analysis and ambient ionization is given. Potential further applications of the DART hair scan method, and ambient ionization in general, are presented, including some preliminary results of new decontamination strategies, hair analysis possibilities, and other forensic uses of DART ionization.