Rapid and simultaneous analysis of multiple drugs in hair samples using dynamic multiple reaction monitoring
Hair analysis for forensic diagnostics is gaining popularity in both research and applied settings. Commercially available dynamic multiple reaction monitoring (Dyn-MRM) software applied to hair samples can provide drug-use history for several months. The cost-effective drug test using Dyn-MRM software facilitates analysis of over 200 analytes in a 10-minute chromatographic run.
by Professor D. P. Naughton and Professor A. Petróczi
Background
Considerable global efforts are expended to address substance abuse which has major effects on public health and quality of life, as well as on economic and societal prosperity. This grand challenge impacts on a wide range of healthcare, regulatory and research endeavours. Key examples include destruction of lives through abuse of class A drugs, efforts to reduce doping in sport, attempts to address alcohol abuse and the lethal dangers of ‘legal highs’ (novel psychoactive substances).
Healthcare and regulatory officials engage in a wide range of activities to combat substance abuse. These include criminalization, banning substances in sport, education programs to prevent and efforts to understand and alter drug-related behaviour. However, in many cases there is an unmitigated failure to address the issues around substance misuse or abuse.
For example, for doping in sports, where vast efforts and resources are expended, indirect assessment of doping produces prevalence figures some 10-fold higher than positive doping test rates [1]. These frequent prevalence reports, at odds with figures from analytical tests, corroborate the belief that current anti-doping testing regimes are far from adequate [1]. Using doping in sport as an exemplar, major improvements in approaches to test for prohibited substances are needed. The advent of more advanced instrumentation aids testing in a number of ways. Increased sensitivity and affordability are very important but so are software developments that provide capability to monitor several hundred substances in one liquid chromatography-tandem mass spectrometry (LC-MS/MS) cycle of less than 10 minutes. These advances bring opportunities that require both instrumentation updates and frequent training updates for staff.
Hair analysis
Despite major advances in instrumentation and software, there are still obstacles to performing successful drug tests that benefit the drug taker. In sport, doping practices are frequently highly advanced, with some athletes taking heed of in-depth knowledge about most key parameters including generic testing methods, masking drugs and advances in detection for specific substances. The burden of proof has shifted to acquiring samples both in and out of competition as well as ensuring that appropriate tests are performed on each sample [2]. Owing to varied pharmacokinetics, analyte distribution and analytical procedures used, testing for a wide range of drugs in all samples is prohibitive [3]. Current testing approaches using biofluids impart considerable practical and financial consequences for the testing regime. Furthermore, cases of microdosing, masking and using novel substances make life challenging for the anti-doping officials.
Drug testing based on biofluids presents a number of issues that add considerably to cost but also are restrictive in terms of the number of tests required to cover a suitable duration owing to the pharmacokinetic profiles of many drugs. Where drugs or their metabolites are washed out efficiently after cessation of use, detection is less viable. The relatively short half-lives of many substances means the window for detection can be limited, which affects the success of occasional testing. The cost of supervised sampling along with the requirement for biofluid storage and handling to avoid sample corruption or infection is prohibitive for major levels of testing. Focusing on doping in sport, further complexities arise through variations in the lists of prohibited substances for testing in and out of competition [4]. The advantage of a longer window of detection via hair-analysis is suited to out of competition testing where a cumbersome system currently exists for sampling which is intrusive and controversial [5]. Thus, new approaches that allow a single test to be conducted simultaneously for (i) a wide range of substances and (ii) covering a prolonged period such as a 3-month window, would be valuable in sport for out of competition testing but also, beyond sport, for social drugs and new psychoactive substances.
In contrast to drug tests based on biofluids, hair analysis provides a range of advantages including: ease of sampling, ability to conduct multiple tests on one cut-hair sample to cover a prolonged duration (a typical 3-cm hair sample is equivalent to approximately 3 months’ growth), lack of issues with infection risk, facile storage at room temperature, lack of requirement to process tissue containing genetic data, and good stability of many drugs and metabolites in the hair matrix.
Instrumentation advances
We recently reported a hair-based method, using liquid chromatography–tandem mass spectrometry (LC-MS/MS), for the analysis of substances of forensic nature [6]. The multi-drug/metabolite assay employs a dynamic multiple reaction monitoring (Dyn-MRM) method using proprietary software [7, 8]. It allows both screening and validated confirmatory analysis depending on the focus of the investigation. This approach has several benefits: (a) the Dyn-MRM software is suited to screen over 200 compounds on a single chromatographic run of under 10 minutes, (b) full validated methods for compounds can be incorporated into the software, (c) hair samples provide the opportunity to cover longer windows for detection in one test (e.g. approximately 3-month history covered in a 3-cm sample), and (d) the software is designed to allow ready adoption of new compounds of interest. The advantage of Dyn-MRM is that multiple reaction monitoring is employed with a focus on scanning for specific peaks at their selected elution times. This efficient method allows the analysis of large numbers of analytes simultaneously in a short run (Fig. 1). In our report, the proprietary software has been extended and applied to cover a range of drugs and metabolites of interest to forensic investigations including cognitive enhancers, amphetamines, barbiturates, benzodiazepines, cannabinoids, cocaine, opioids, steroids and sedatives. The chromatographic run is calibrated by a test mixture containing approximately 20 substances and further tailoring would be required to match a specific remit such as the WADA (World Anti-Doping Agency) prohibited list more closely [4].
Conclusion and future perspectives
The application of Dyn-MRM software to screen for a large range of drugs brings considerable advantages to laboratories involved in drug testing. The ease of use and ability to add new compounds to the screening database are noteworthy. Coupling this commercially available software to hair analysis adds the extra dimension of being able to screen for drug use over several months in one hair sample. This advance will add considerably to the efficiency of drug testing but will remain as an adjunct to other testing methods for out of competition testing in sport as it will not cover all analytes of interest to anti-doping officials [4]. Some substances are unlikely to be found in hair (e.g. performance enhancing peptides) and for other substances there will be issues with establishing a threshold – either for endogenous substances (e.g. testosterone) or for substances consumed through diet (e.g. drugs used in farming). Further limitations are that (i) a single use of a drug may be undetectable owing to the low levels deposited in hair, and (ii) more research is warranted to ascertain the effects of hair type and colour on analyte uptake and stability. In spite of these limitations, hair analysis coupled to modern advances in instrumentation sensitivity and software capabilities is promising in many scenarios especially to obtain a prolonged history of abuse and where ‘zero tolerance’ is applied (e.g. for synthetic steroids). In addition, hair analysis may have a role in support of the Athlete Biological Passport through analysis of indirect biomarkers of doping [9].
References
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2. World Anti-Doping Agency (WADA). International Standards. 2015; https://www.wada-ama.org/en/what-we-do/international-standards.
3. Maennig W. Inefficiency of the anti-doping system: Cost reduction proposals. Subst Use Misuse 2014; 49(9): 1201–1205.
4. WADA. List of prohibited substances and methods. 2015; http://list.wada-ama.org/.
5. Hanstad DV, Loland S. Elite athletes’ duty to provide information on their whereabouts: Justifiable anti-doping work or an indefensible surveillance regime? Eur J Sport Sci. 2009; 9(1): 3–10.
6. Shah I, Petroczi A, Uvacsek M, Ranky M, Naughton DP. Hair-based rapid analyses for multiple drugs in forensics and doping: application of dynamic multiple reaction monitoring with LC-MS/MS. Chem Cent J. 2014; 8(1): 73.
7. Agilent Technical Overview. Ion optics innovations for increased sensitivity in hybrid MS systems. Agilent Technologies USA 5989-7408EN. 2007; http://www.chem.agilent.com/Library/technicaloverviews/Public/5989-7408EN_HI.pdf.
8. Stone P, Glauner T, Kuhlmann F, Schlabach Tim, Miller K. New dynamic MRM mode improves data quality and triple quad quantification in complex analyses. Agilent Technologies USA 5990-3595EN. 2009; http://www.chem.agilent.com/Library/technicaloverviews/Public/5990-3595en_lo%20CMS.pdf.
9. Vernec AR. The athlete biological passport: an integral element of innovative strategies in antidoping. Br J Sports Med. 2014; 48(10):817–819.
The authors
Declan P. Naughton* PhD, Andrea Petróczi PhD
School of Life Sciences, Kingston University, London, UK
*Corresponding author
E-mail: D.Naughton@kingston.ac.uk
