Matrix-assisted laser-desorption/ionization mass spectrometric imaging of olanzapine in a single hair using esculetin as a matrix

Highlights

• MALDI-MS imaging of olanzapine in hair using esculetin as a matrix.

• Esculetin increased the affinity, extraction and ionization efficiency of olanzapine.

• The spatial distributions of olanzapine were observed throughout hairs from drug users.

• MALDI imaging intensities showed good semi-quantitative correlation to HPLC–MS/MS.

• MALDI-MSI is suitable for monitoring drug of intake with a high time resolution.

Abstract

Matrix-assisted laser desorption/ionization-mass spectrometric imaging (MALDI-MSI) for the analysis of intact hair is a powerful tool for monitoring changes in drug consumption. The embedding of a low drug concentration in the hydrophobic hair matrix makes it difficult to extract and detect, and requires an improved method to increase detection sensitivity. In this study, an MSI method using MALDI-Fourier transform ion cyclotron resonance was developed for direct identification and imaging of olanzapine in hair samples using the positive ion mode. Following decontamination, scalp hair samples from an olanzapine user were scraped from the proximal to the distal end three times, and 5 mm hair sections were fixed onto an Indium-Tin-Oxide (ITO)-coated microscopic glass slide. Esculetin (6,7-dihydroxy-2H-chromen-2-one) was used as a new hydrophobic matrix to increase the affinity, extraction and ionization efficiency of olanzapine in the hair samples. The spatial distribution of olanzapine was observed using five single hairs from the same drug user. This matrix improves the affinity of olanzapine in hair for molecular imaging with mass spectrometry. This method may provide a detection power for olanzapine to the nanogram level per 5 mm hair. Time course changes in the MSI results were also compared with quantitative HPLC–MS/MS for each 5 mm segment of single hair shafts selected from the MALDI target. MALDI imaging intensities in single hairs showed good semi-quantitative correlation with the results from conventional HPLC–MS/MS. MALDI-MSI is suitable for monitoring drug intake with a high time resolution.

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.