Matrix-assisted laser-desorption/ionization mass spectrometric imaging of olanzapine in a single hair using esculetin as a matrix
Graphical abstract
Introduction
Hair analysis provides useful information on the history and time course of drug intake in forensic science and medicine. Gas chromatography–mass spectrometry (GC–MS) [1] and liquid chromatography–mass spectrometry (LC–MS) [2] are usually used to detect drugs and their metabolites in hair samples. A series of hair segments from the root to the tip were analyzed and provided historic information on drug intake, but the resolution was limited. As time-consuming and tedious pretreatment steps are usually required prior to analysis, including washing, pulverization, digestion under acid or alkaline conditions, liquid–liquid extraction or solid-phase extraction, and evaporation under nitrogen gas or vacuum, it is necessary to develop a direct and simple analytical technique to detect target drugs in hair samples. Matrix-assisted laser desorption/ionization (MALDI)-coupled with mass spectrometric imaging (MSI) is a rapidly emerging technology that can reflect the distribution of small pharmaceutical molecules and their metabolites in tissue sections [3], [4], [5], [6], [7], [8], [9], [10], [11], [12], plants [13], [14], [15], [16], [17] and pharmaceutical tablets [18]. MALDI-MSI can display the spatial localization and distribution of drugs during segmental hair analysis, and can also provide useful information regarding the state and history of drug use in medicinal research, therapeutic monitoring and forensic applications. A few investigations have reported the outcome of MALDI-MSI of psychotropic drugs in a single hair and following careful optimization of sample preparation, target drugs including methamphetamine [19], ketamine [20], zolpidem [21], methoxyphenamine [22], tilidine [23], cocaine [24], [25], cannabinoids [26] and efavirenz [27] have been evaluated.
However, compared with tissue sections, hair is a keratinous fiber, and is strongly hydrophobic. A number of psychotropic drugs such as olanzapine are also hydrophobic, thus cannot be completely dissolved in water. Conventional MALDI matrices, such as 2,5-dihydroxybenzoic acid (DHB) and a-cyano-4-hydroxycinnamic acid (CHCA) have hydrophilic properties [28], and thus may influence the detection and affinity of hydrophobic compounds in the hair sample. In addition, the nonhomogeneous crystallization and large crystal size of DHB can cause molecular delocalization and poor spot-to-spot reproducibility (modern application methods such as sublimation can provide crystal sizes that are substantially smaller than the width of the hair). Moreover, the nanogram per milligram level of drug and embedding of the drug in the hydrophobic hair matrix require higher detection sensitivity using MALDI-MSI. In order to increase the detection limit of hydrophobic compounds and homogeneous crystallization, alkylated trihydroxyacetophenone and alkylated DHB have been developed as MALDI matrices for the analysis of hydrophobic peptides [28]. 6-Aza-2-thiothymine and 4-phenyl-a-cyanocinnamic acid have been used to detect oxidized phospholipids and lipids, respectively [29], [30]. However, a satisfactory and optimized MALDI matrix for the enrichment and analysis of hydrophobic samples such as hair is still needed with high affinity for hydrophobic drugs in hair samples.
In the present study, olanzapine was chosen as example of hydrophobic substance among psychotropic drugs, esculetin (6,7-dihydroxy-2H-chromen-2-one) was used as a hydrophobic matrix for MALDI-FT ICR MSI of olanzapine in longitudinal sections of single hair shafts obtained from an olanzapine user. Single scalp hair samples were scraped manually and coated with esculetin matrix. The MALDI-FT ICR MSI method was developed for the identification, semi-quantification and imaging of olanzapine in five hair samples approximately 5 mm in length. In addition, the conventional HPLC–MS/MS method for absolute quantification was performed and compared with the semi-or relative quantitative results of the MALDI-MSI method. Time-course changes in imaging results were supported by confirmatory quantitative HPLC–MS/MS analysis. The results provides useful information on the history and time course of drug intake in clinical and forensic research. By measuring the length of the hair and approximating the ratio of hair growth, it is possible to estimate when drug intake occurred. The analytical method using esculetin may be also extended to MALDI-MS imaging of other psychoactive hydrophobic substances.
Section snippets
Materials and chemicals
Olanzapine standard (99.9%), LC–MS grade acetonitrile, formic acid, ethanol and methanol were provided by Anpel Laboratory Technologies (Shanghai, China). DHB and CHCA were purchased from Sigma Chemical Co. (St. Louis, MO, USA); the deionized water used in this study was obtained using a Milli-Q water purification system (Millipore Corp., Bedford, MA, USA); esculetin was bought from TCI Chemical Co. (Tokyo, Japan). 1.0 mg mL−1 methanol solution of d8-olanzapine was purchased from Cambridge
Sample preparation for MALDI imaging
The choice of matrix and matrix deposition methods are critical factors determining the properties of the matrix layer and the spatial resolution. Various matrix deposition methods have been developed including manual and automatic deposition such as the use of an airbrush, sprayer, inkjet printer, and sublimation. As drugs are entrapped mainly in the core of the hair shaft, sensitivity was very poor when the intact hair was coated and micro-spotted directly. In order to solve this problem,
Conclusions
This study demonstrates that esculetin is a useful matrix for the analysis of olanzapine in longitudinal sections of hair by MALDI-FT ICR MS in positive-ion mode. MALDI-MSI using esculetin has great potential in the analysis of olanzapine in a single hair shaft. MALDI-MSI single segmental hair analysis is suitable for monitoring and comparing drugs with high time resolution and may have useful applications in medicine.
Conflict of interest
All authors declare that they have no conflict of interest.
Acknowledgments
This work was supported by the Fund from Shanghai Key Laboratory of Forensic Medicine (Department of Forensic Toxicology, Institute of Forensic Sciences, Ministry of Justice) (grant No KF1602), the National Natural Science Foundation of China (grant No. 21405104); and the Youth Initial Foundation of Shanghai Jiao Tong University.
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Interpol review of toxicology 2016–2019
2020, Forensic Science International: SynergyCitation Excerpt :In the articles, the sample preparation and the instrumental parameters, such as spatial resolution, raster speed and sample orientation have been discussed. Most of the IMS studies on hair analysis used MALDI-MS, such as the mapping of cannabinoids in single hair [536], characterization of synthetic cannabinoid isomers in single hair [537], olanzapines with esculetin as matrix [538], analysis of methamphetamine with umbelliferone as a matrix [539], and the study of zolpidem distribution in hair after a single administration [540]. Instead of MALDI, another ionization technique, DART (direct analysis in real time) was used in IMS study in forensic hair analysis [541].
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