Justin M. Wiseman

Ambient Mass Spectrometry

A recent innovation in mass spectrometry is the ability to record mass spectra on ordinary samples, in their native environment, without sample preparation or preseparation by creating ions outside the instrument. In desorption electrospray ionization (DESI), the principal method described here, electrically charged droplets are directed at the ambient object of interest; they release ions from the surface, which are then vacuumed through the air into a conventional mass spectrometer. Extremely rapid analysis is coupled with high sensitivity and high chemical specificity. These characteristics are advantageously applied to high-throughput metabolomics, explosives detection, natural products discovery, and biological tissue imaging, among other applications. Future possible uses of DESI for in vivo clinical analysis and its adaptation to portable mass spectrometers are described.

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.

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.

Ambient molecular imaging by desorption electrospray ionization mass spectrometry

Desorption electrospray ionization (DESI) allows the direct analysis of ordinary objects or pre-processed samples under ambient conditions. Among other applications, DESI is used to identify and record spatial distributions of lipids and drug molecules in biological tissue sections. This technique does not require sample preparation other than production of microtome tissue slices and does not involve the use of ionization matrices. This greatly simplifies the procedure and prevents the redistribution of analytes during matrix deposition. Images are obtained by continuously moving the sample relative to the DESI sprayer and the inlet of the mass spectrometer. The timing of the protocol depends on the size of the surface to be analyzed and on the desired resolution. Analysis of organ tissue slices at 250 μm resolution typically takes between 30 min and 2 h.

Direct analysis of dried blood spots utilizing desorption electrospray ionization (DESI) mass spectrometry

A novel approach to the quantitative determination of xenobiotics in whole blood samples without sample preparation or chromatography is described. This method is based on direct analysis of microlitre volumes of blood which are spotted onto specialized paper cards and dried, with the resulting dried blood spots (DBS) analyzed directly via desorption electrospray ionization (DESI) mass spectrometry (MS). Using sitamaquine, terfenadine, and prazosin as model compounds with verapamil as a common internal standard, this methodology demonstrated detection of each compound down to 10 ng mL(-1) from DBS where standard calibration curves show linearity from 10-10,000 ng mL(-1) with r(2) > 0.99. Three (3) different untreated types of filter papers (Whatman 903 and 31ETF as well as Ahlstrom 237) and two (2) treated types of filter paper (Whatman FTA and FTA Elute) were examined and the effect of each surface on the recovery of each analyte was evaluated. The results show that the untreated papers provide the best substrates for DBS analysis by DESI. A more in depth study of the quantitation of sitamaquine on 31ETF paper stock provided bias and error measurements of less than 20%. The promising results shown in this study may have important implications in the areas of therapeutic drug monitoring (TDM), clinical and forensic toxicology, and pharmacology.

Direct analysis of Salvia divinorum leaves for salvinorin A by thin layer chromatography and desorption electrospray ionization multi‐stage tandem mass spectrometry

Salvia divinorum is widely cultivated in the US, Mexico, Central and South America and Europe and is consumed for its ability to produce hallucinogenic effects similar to those of other scheduled hallucinogenic drugs, such as LSD. Salvinorin A (SA), a kappa opiod receptor agonist and psychoactive constituent, is found primarily in the leaves and to a lesser extent in the stems of the plant. Herein, the analysis of intact S. divinorum leaves for SA and of acetone extracts separated using thin layer chromatography (TLC) is demonstrated using desorption electrospray ionization (DESI) mass spectrometry. The detection of SA using DESI in the positive ion mode is characterized by several ions associated with the compound - [M+H](+), [M+NH(4)](+), [M+Na](+), [2M+NH(4)](+), and [2M+Na](+). Confirmation of the identity of these ions is provided through exact mass measurements using a time-of-flight (ToF) mass spectrometer. The presence of SA in the leaves was confirmed by multi-stage tandem mass spectrometry (MS(n)) of the [M+H](+) ion using a linear ion trap mass spectrometer. Direct analysis of the leaves revealed several species of salvinorin in addition to SA as confirmed by MS(n), including salvinorin B, C, D/E, and divinatorin B. Further, the results from DESI imaging of a TLC separation of a commercial leaf extract and an acetone extract of S. divinorum leaves were in concordance with the TLC/DESI-MS results of an authentic salvinorin A standard. The present study provides an example of both the direct analysis of intact plant materials for screening illicit substances and the coupling of TLC and DESI-MS as a simple method for the examination of natural products.

Development of Stereotactic Mass Spectrometry for Brain Tumor Surgery

BACKGROUND: Surgery remains the first and most important treatment modality for the majority of solid tumors. Across a range of brain tumor types and grades, postoperative residual tumor has a great impact on prognosis. The principal challenge and objective of neurosurgical intervention is therefore to maximize tumor resection while minimizing the potential for neurological deficit by preserving critical tissue. OBJECTIVE: To introduce the integration of desorption electrospray ionization mass spectrometry into surgery for in vivo molecular tissue characterization and intraoperative definition of tumor boundaries without systemic injection of contrast agents. METHODS: Using a frameless stereotactic sampling approach and by integrating a 3-dimensional navigation system with an ultrasonic surgical probe, we obtained image-registered surgical specimens. The samples were analyzed with ambient desorption/ionization mass spectrometry and validated against standard histopathology. This new approach will enable neurosurgeons to detect tumor infiltration of the normal brain intraoperatively with mass spectrometry and to obtain spatially resolved molecular tissue characterization without any exogenous agent and with high sensitivity and specificity. RESULTS: Proof of concept is presented in using mass spectrometry intraoperatively for real-time measurement of molecular structure and using that tissue characterization method to detect tumor boundaries. Multiple sampling sites within the tumor mass were defined for a patient with a recurrent left frontal oligodendroglioma, World Health Organization grade II with chromosome 1p/19q codeletion, and mass spectrometry data indicated a correlation between lipid constitution and tumor cell prevalence. CONCLUSION: The mass spectrometry measurements reflect a complex molecular structure and are integrated with frameless stereotaxy and imaging, providing 3-dimensional molecular imaging without systemic injection of any agents, which can be implemented for surgical margins delineation of any organ and with a rapidity that allows real-time analysis.

Coupling Desorption Electrospray Ionization with Solid-Phase Microextraction for Screening and Quantitative Analysis of Drugs in Urine

Direct analysis of silica C(18)-coated solid-phase microextraction (SPME) fibers using desorption electrospray ionization mass spectrometry (DESI-MS) for the purpose of analyzing drugs from raw urine is presented. The method combines a simple, inexpensive, and solvent-less sample preparation technique with the specificity and speed of DESI-MS and MS/MS. Extraction of seven drugs from raw urine is performed using specially designed SPME fibers coated uniformly with silica-C(18) stationary phase. Each SPME device is inserted into unprocessed urine under gentle agitation and, then, removed, rinsed, and analyzed directly by DESI-MS (MS/MS). Rapid screening over a wide mass range is afforded by coupling the method with a time of flight (TOF) mass spectrometer while quantitative analysis is performed using selected reaction monitoring (SRM) using a triple quadrupole mass spectrometer. The performance of the SPME DESI-MS/MS method was evaluated by preparing calibration standards and quality control (QC) samples of the seven drug compounds from urine over a range from 20 to 1000 ng/mL, with the exception of meprobamate which was prepared from 200 to 10000 ng/mL. The calibration curves constructed for each analyte had an R(2) > 0.99. The range of precision (%CV) and accuracy values (% bias) for low QC samples was 1-11% and 3-38%, respectively. Precision and accuracy values for high QC samples range from 0.9 to 8% and -31 to -8%. Results from urine specimens of actual exposure to drugs screened using the SPME DESI-MS/MS method showed good agreement with the conventional immunoassays and GC/MS analysis. Liquid desorption of the SPME fiber followed by LC/MS/MS also showed good agreement with the SPME DESI-MS/MS method.

Evaluation and performance of desorption electrospray ionization using a triple quadrupole mass spectrometer for quantitation of pharmaceuticals in plasma

The present work describes the methodology and investigates the performance of desorption electrospray ionization (DESI) combined with a triple quadrupole mass spectrometer for the quantitation of small drug molecules in human plasma. Amoxepine, atenolol, carbamazepine, clozapine, prazosin, propranolol and verapamil were selected as target analytes while terfenadine was selected as the internal standard common to each of the analytes. Protein precipitation of human plasma using acetonitrile was utilized for all samples. Limits of detection were determined for all analytes in plasma and shown to be in the range 0.2-40 ng/mL. Quantitative analysis of amoxepine, prazosin and verapamil was performed over the range 20-7400 ng/mL and shown to be linear in all cases with R(2) >0.99. In most cases, the precision (relative standard deviation) and accuracy (relative error) of each method were less than or equal to 20%, respectively. The performance of the combined techniques made it possible to analyze each sample in 15 s illustrating DESI tandem mass spectrometry (MS/MS) as powerful tool for the quantitation of analytes in deproteinized human plasma.

Data Processing for 3D Mass Spectrometry Imaging

Data processing for three dimensional mass spectrometry (3D-MS) imaging was investigated, starting with a consideration of the challenges in its practical implementation using a series of sections of a tissue volume. The technical issues related to data reduction, 2D imaging data alignment, 3D visualization, and statistical data analysis were identified. Software solutions for these tasks were developed using functions in MATLAB. Peak detection and peak alignment were applied to reduce the data size, while retaining the mass accuracy. The main morphologic features of tissue sections were extracted using a classification method for data alignment. Data insertion was performed to construct a 3D data set with spectral information that can be used for generating 3D views and for data analysis. The imaging data previously obtained for a mouse brain using desorption electrospray ionization mass spectrometry (DESI-MS) imaging have been used to test and demonstrate the new methodology.