Demian R. Ifa

Desorption electrospray ionization mass spectrometry: Imaging drugs and metabolites in tissues

Ambient ionization methods for MS enable direct, high-throughput measurements of samples in the open air. Here, we report on one such method, desorption electrospray ionization (DESI), which is coupled to a linear ion trap mass spectrometer and used to record the spatial intensity distribution of a drug directly from histological sections of brain, lung, kidney, and testis without prior chemical treatment. DESI imaging provided identification and distribution of clozapine after an oral dose of 50 mg/kg by: i) measuring the abundance of the intact ion at m/z 327.1, and ii) monitoring the dissociation of the protonated drug compound at m/z 327.1 to its dominant product ion at m/z 270.1. In lung tissues, DESI imaging was performed in the full-scan mode over an m/z range of 200-1100, providing an opportunity for relative quantitation by using an endogenous lipid to normalize the signal response of clozapine. The presence of clozapine was detected in all tissue types, whereas the presence of the N-desmethyl metabolite was detected only in the lung sections. Quantitation of clozapine from the brain, lung, kidney, and testis, by using LC-MS/MS, revealed concentrations ranging from 0.05 μg/g (brain) to a high of 10.6 μg/g (lung). Comparisons of the results recorded by DESI with those by LC-MS/MS show good agreement and are favorable for the use of DESI imaging in drug and metabolite detection directly from biological tissues.

Latent Fingerprint Chemical Imaging by Mass Spectrometry

Latent fingerprints (LFPs) potentially contain more forensic information than the simple identification of the subject; they may contain evidence of contacts with explosives or substances of abuse. Chemical information can also be useful in resolving overlapping LFPs from different individuals. We used desorption electrospray ionization mass spectrometry in an imaging mode to record compound-specific chemical fingerprints.

Mass Spectrometry Imaging under Ambient Conditions

Mass spectrometry imaging (MSI) has emerged as an important tool in the last decade and it is beginning to show potential to provide new information in many fields owing to its unique ability to acquire molecularly specific images and to provide multiplexed information, without the need for labeling or staining. In MSI, the chemical identity of molecules present on a surface is investigated as a function of spatial distribution. In addition to now standard methods involving MSI in vacuum, recently developed ambient ionization techniques allow MSI to be performed under atmospheric pressure on untreated samples outside the mass spectrometer. Here we review recent developments and applications of MSI emphasizing the ambient ionization techniques of desorption electrospray ionization (DESI), laser ablation electrospray ionization (LAESI), probe electrospray ionization (PESI), desorption atmospheric pressure photoionization (DAPPI), femtosecond laser desorption ionization (fs-LDI), laser electrospray mass spectrometry (LEMS), infrared laser ablation metastable-induced chemical ionization (IR-LAMICI), liquid microjunction surface sampling probe mass spectrometry (LMJ-SSP MS), nanospray desorption electrospray ionization (nano-DESI), and plasma sources such as the low temperature plasma (LTP) probe and laser ablation coupled to flowing atmospheric-pressure afterglow (LA-FAPA). Included are discussions of some of the features of ambient MSI for example the ability to implement chemical reactions with the goal of providing high abundance ions characteristic of specific compounds of interest and the use of tandem mass spectrometry to either map the distribution of targeted molecules with high specificity or to provide additional MS information on the structural identification of compounds. We also describe the role of bioinformatics in acquiring and interpreting the chemical and spatial information obtained through MSI, especially in biological applications for tissue diagnostic purposes. Finally, we discuss the challenges in ambient MSI and include perspectives on the future of the field.

Rapid, Direct Analysis of Cholesterol by Charge Labeling in Reactive Desorption Electrospray Ionization

Direct and rapid analysis of cholesterol was accomplished in the ambient environment using reactive desorption electrospray ionization (DESI) mass spectrometry. This was achieved by electrospraying reagent solutions in the form of high velocity charged droplets at surfaces such as dried serum samples and animal tissue sections. Betaine aldehyde, incorporated into the spray solvent, reacts selectively and rapidly with the alcohol group of cholesterol by nucleophilic addition, forming a hemiacetal salt. Limits of detection for pure cholesterol and related compounds were approximately 1 ng when a solution of cholesterol of 1 microg/mL was spotted onto the surface. Quantitative analysis of free cholesterol in serum using reactive DESI was demonstrated using cholesterol-d7 as internal standard. High throughput analysis of small volumes of serum spotted onto a suitable substrate was achieved at an analysis rate of approximately 14 s per sample, with a relative standard deviation (RSD) of ca. 6%. Use of reactive DESI in the imaging mode allowed 2D spatial distributions of phospholipids and cholesterol to be recorded simultaneously in rat brain tissues.

Forensic analysis of inks by imaging desorption electrospray ionization (DESI) mass spectrometry

Desorption electrospray ionization mass spectrometry (DESI-MS) is employed in the forensic analysis of documents. Blue ballpoint pen inks applied to ordinary writing paper are examined under ambient conditions without any prior sample preparation. When coupled to an automated moving stage, two-dimensional molecular images are generated. Proof-of-principle experiments include characterization of a simulated forged number and examination of older written records. This application of DESI has advantages over extractive techniques in terms of speed and sample preservation. The effects of the desorbing solvent composition, in this case a mixture of methanol and water, and of flow rate, are evaluated. Results suggest that the solubility of the analyte (dyes Basic Blue 7, Basic Violet 3 and Solvent Blue 26) plays an important role in desorption from the paper surface.

Forensic applications of ambient ionization mass spectrometry

This review highlights and critically assesses forensic applications in the developing field of ambient ionization mass spectrometry. Ambient ionization methods permit the ionization of samples outside the mass spectrometer in the ordinary atmosphere, with minimal sample preparation. Several ambient ionization methods have been created since 2004 and they utilize different mechanisms to create ions for mass-spectrometric analysis. Forensic applications of these techniques—to the analysis of toxic industrial compounds, chemical warfare agents, illicit drugs and formulations, explosives, foodstuff, inks, fingerprints, and skin—are reviewed. The minimal sample pretreatment needed is illustrated with examples of analysis from complex matrices (e.g., food) on various substrates (e.g., paper). The low limits of detection achieved by most of the ambient ionization methods for compounds of forensic interest readily offer qualitative confirmation of chemical identity; in some cases quantitative data are also available. The forensic applications of ambient ionization methods are a growing research field and there are still many types of applications which remain to be explored, particularly those involving on-site analysis. Aspects of ambient ionization currently undergoing rapid development include molecular imaging and increased detection specificity through simultaneous chemical reaction and ionization by addition of appropriate chemical reagents.

Desorption electrospray ionization (DESI) mass spectrometry and tandem mass spectrometry (MS/MS) of phospholipids and sphingolipids: Ionization, adduct formation, and fragmentation

Desorption electrospray ionization (DESI) mass spectrometry was evaluated for the characterization of glycerophospholipid standards, including glycerophosphocholine (GPCho), glycerophosphoglycerol (GPGro), glycerophosphoethanolamine (GPEtn), glycerophosphoserine (GPSer), glycerophosphoinositol (GPIns), cardiolipin (CL), and sphingolipid standards, including sulfatides (ST) and sphingomyelin (SM). Of specific interest were the effects of surface and solvent composition on signal stability and intensity, along with the ions observed in the full scan mode and the fragmentations seen upon collisional activation for each of the above classes. These experiments were performed without the addition of matrix compounds to the sample and were conducted in the free ambient environment at atmospheric pressure. The compounds GPSer, GPGro, GPIns, ST, and CL were best analyzed in the negative ion mode while PE was ionized efficiently in both positive and negative ion modes. SM and GPCho, which typically generate more abundant ions in the positive ion mode, could be analyzed in the negative ion mode by the addition of anionic reagents such as acetate to the spray solvent. Full scan DESI mass spectra and tandem (MS/MS) spectra for this representative set of physiological phospho/sphingolipids are presented. Similarities with other ionization methods in terms of fragmentation behavior were strong, although ambient ionization of untreated samples is only available with DESI. The effect of surface and solvent properties on signal intensity and stability were determined by depositing standard compounds on several different surfaces and analyzing with various proportions of methanol in the aqueous spray. Analysis was extended to complex mixtures of phospholipids and sphingolipids by examining the total lipid extract of porcine brain and by direct analysis of rat brain cryotome sections. These types of mixture analyses and molecular imaging studies are likely to represent major areas of application of DESI.

Three‐Dimensional Vizualization of Mouse Brain by Lipid Analysis Using Ambient Ionization Mass Spectrometry

Two-dimensional (2D) imaging mass spectrometry (MS)[1] has emerged as a powerful technique in the biological sciences. It allows direct investigation of the distribution of a variety of lipids, drugs, biological defensive agents, pigments and proteins in plant and animal tissues with high specificity and without the need of fluorescent or radioactive labelling normally used in histochemical protocols.[2, 3] In imaging MS, the chemical identity of molecules present on a surface is investigated as a function of their 2D spatial distribution (x and y coordinates).

Cholesterol Sulfate Imaging in Human Prostate Cancer Tissue by Desorption Electrospray Ionization Mass Spectrometry

Development of methods for rapid distinction between cancerous and non-neoplastic tissues is an important goal in disease diagnosis. To this end, desorption electrospray ionization mass spectrometry (DESI-MS) imaging was applied to analyze the lipid profiles of thin tissue sections of 68 samples of human prostate cancer and normal tissue. The disease state of the tissue sections was determined by independent histopathological examination. Cholesterol sulfate was identified as a differentiating compound, found almost exclusively in cancerous tissues including tissue containing precancerous lesions. The presence of cholesterol sulfate in prostate tissues might serve as a tool for prostate cancer diagnosis although confirmation through larger and more diverse cohorts and correlations with clinical outcome data is needed.

Desorption Electrospray Ionization Mass Spectrometry for Lipid Characterization and Biological Tissue Imaging

Desorption electrospray ionization mass spectrometry (DESI-MS) imaging of biological samples allows untargeted analysis and structural characterization of lipids ionized from the near-surface region of a sample under ambient conditions. DESI is a powerful and sensitive MS ionization method for 2D and 3D imaging of lipids from direct and unmodified complex biological samples. This review describes the strengths and limitations of DESI-MS for lipid characterization and imaging together with the technical workflow and a survey of applications. Included are discussions of lipid mapping and biomarker discovery as well as a perspective on the future of DESI imaging.