A simple and robust method for broad range screening of hair samples for drugs of abuse using a high-throughput UHPLC-Ion Trap MS instrument
Introduction
It is beyond of any doubts that the possibility of performing a wide spectrum search for drugs and toxicants is a fundamental condition for any correct diagnostic approach in both clinical and forensic medicine. Traditionally, this problem has been faced using an approach known as “toxicological screening” usually performed by immunoassays. In fact, this technology is known to provide high productivity at reasonable costs without requiring skilled personnel. Unfortunately, it is characterized by moderate specificity and sensitivity and by a quite narrow panel of identifiable compounds. If traditionally the typical biological samples used in clinical and forensic toxicology were urine and blood (and gastric content, as well as autopsy specimens), more recently other biological matrices, e.g. hair, oral fluid, sweat, and vitreous humour, have proved to provide important information for a correct diagnosis. Among these alternative biological matrices, hair has found wide interest and acceptance as it is known to provide evidence on the history of drug intake of a subject, extending for several months before hair collection [1]. In this particular context, considering the small amounts of the sample which can be collected, the needs of analytical sensitivity are crucial, and for this reason, only the most sensitive immunoassays could be applied (e.g. in the past, radioimmunoassays and, recently, enzyme-linked immunosorbent assays) [1]. Consequently, because of a limited availability of antisera with adequate specificity, this technology has successfully been used only for the presumptive identification of a limited number of analytes, including opiates, cocaine and metabolites, THC and metabolites, amphetamines, barbiturates, benzodiazepines and few other. Considering the recent increase of abused and misused drugs, spanning from the traditional “NIDA FIVE” (i.e. marijuana, phencyclidine, opiates, amphetamines, and cocaine) to the recent New Psychoactive Substances (e.g. synthetic cannabinoids, cathinones, fentanyls and new benzodiazepines) and pharmaceuticals (e.g. sleep inducers, analgesics and sedatives) the diagnostic sensitivity of a drug screening merely based on immunological techniques looks today largely insufficient. This flaw is particularly relevant if one considers that a “negative” result at the screening is directly reported not requiring any other further confirmation by alternative techniques, which is instead requested after a “positive” result. For this reason, in the screening phase, the diagnostic sensitivity, i.e. the ability to identify all “positive” cases in a population, is crucial.
As it is well-known, in the last decades, alternative approaches to the toxicological screenings using different techniques of mass spectrometry have been extensively applied, not only for blood and urine analysis but also for hair testing [1].
In this context, either GC or LC separation techniques have been used generally coupled to single and triple quadrupoles, and more recently to high-resolution mass spectrometers. In particular, in the early times of hair analysis [2], GC-single quadrupole-MS was the first instrumental screening technique adopted [3], [4] and still today it is largely used being affordable, robust and well-established in terms of hardware, software and databases. However, recent technological improvements regarding LC-MS instruments permitted to show neat advantages, particularly in terms of sensitivity, allowing to simplify the procedures for sampling preparation (i.e. dilute-and-shoot injection). The MS equipment generally used for this purpose includes low-resolution mass spectrometers (LRMS), in general using triple quadrupole analyzers, and high-resolution mass spectrometers (HRMS). Although, both LRMS and HRMS are currently used [5], each one of the two technologies show distinct weak points. In particular, the former requires an a priori knowledge of the target analytes showing a progressive loss of sensitivity proportional to the number of targets which are monitored simultaneously. HRMS, on the other hand, is characterized by higher costs, lower performance in quantitative analyses and, although not limited by the need of a list of the target analytes, its application in practice requires highly skilled personnel [5]. This limitation hampers the adoption of LC-MS in non-specialized analytical contexts, such as in clinical chemistry and toxicology laboratories where most of the toxicological screenings are carried out.
To the best of our knowledge, very few LC-MS platforms have been developed specifically for a “user-friendly” toxicological screening, being the majority of them dedicated to targeted quantitative analysis of the common drugs of abuse. In 2014, the first application of an interesting LC-MS instrumentation fitted with a user-friendly software dedicated to drug screening was reported by Kempf et al. [6]. This approach was based on UHPLC separation and ESI-LRMS identification using a high speed three dimensional Ion Trap (IT) with MS2 and MS3 capability (Toxtyper®, Bruker Daltonics, Bremen, Germany). This equipment, reportedly, was designed to be used by MS-unskilled personnel in heavy routine contexts. A simplified user-interface with a push-button approach is associated with validated chromatographic and MS conditions and includes an open MS spectra database [7]. In recent years, the Toxtyper® has been tested mainly for urine [8], [9] and blood [6], [10] analysis. Other reported applications include oral fluid [11], vitreous humour [12] and dental hard tissue [13].
The aim of the present work was to verify the usefulness of this simplified LC-MS procedure for the toxicological screening of hair in a routine setting, using as benchmark the standard protocol in use in our laboratories including LC-MS and GC–MS [14]. In order to face the needs of a toxicological screening, it was chosen to avoid any sample pretreatment after the extraction of the drugs from the hair matrix by classical incubation in acid medium, which was neutralized before direct injection.
Undoubtedly, another crucial feature of the screening methods, in addition to the simplicity of the pre-analytical procedures, is sensitivity, since only “positive” samples at the toxicological screening undergo further confirmation with a more specific and quantitative technology. In order to optimize this feature of Toxtyper® two different ion sources were tested, one of which follows a typical ESI scheme, while the other (ionBooster™, Bruker) is a proprietary form of heated-ESI, reportedly able to increase the ionization of the analytes.
Section snippets
Chemicals
All chemicals were of analytical reagent grade. Formic acid, ammonium formate, hydrochloric acid, 2-propanol, HPLC grade acetonitrile and HPLC grade methanol were purchased from Sigma-Aldrich (Darmstadt, Germany).
Amphetamine (A), metamphetamine (MA), 3,4-methylenedioxyamphetamine (MDA), 3,4-methylenedioxymethamphetamine (MDMA), 1,3-benzodioxolyl-N-methylbutanamine (MBDB), 3,4- methylenedioxy-N-ethylamphetamine (MDEA), norketamine (NK), ketamine (K) and scopolamine were purchased from LGC
Results and discussion
The present work was aimed at the evaluation of the application to hair testing of a simplified push-button approach to toxicological analysis based on the use of an LC-Ion Trap MS platform (ToxTyper®). Being the Toxtyper® an analytical tool dedicated to the toxicological screening of biological fluids, it looked necessary a specific validation for analyzing a peculiar biological matrix, such as hair.
Conclusions
The present work reports the analytical performance of a simple LC-MS approach to the qualitative toxicological screening applied to hair analysis in a routine context. Sensitive and reliable analytical information was produced by the tested instrument using an external confirmation with a validated GC–MS procedure on a panel of 16 drugs of abuse and metabolites. Also, the accuracy of this analytical tool was verified by analyzing proficiency test samples (N = 12) containing not only
Funding
This work was supported by the Fondazione Cariverona [grant number CUP B36J16002570003]
CRediT authorship contribution statement
Giacomo Musile: Conceptualization, Methodology, Writing - original draft. Mara Mazzola: Investigation. Ksenia Shestakova: Investigation. Sergey Savchuk: Supervision. Svetlana Appolonova: Supervision. Franco Tagliaro: Funding acquisition, Writing - review & editing.
Declaration of Competing Interest
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
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