Rapid analysis of fentanyls and other novel psychoactive substances in substance use disorder patient urine using paper spray mass spectrometry.

RATIONALE: Drug overdose deaths due to fentanyls and other novel psychoactive substances (NPS) are on the rise. The higher potencies of fentanyl analogs compared with morphine require new technologies to identify and quantitate NPS. METHODS: Paper spray tandem mass spectrometry (MS/MS) and high-resolution mass spectrometry were used to identify and measure fentanyl analogs as well as common drugs of abuse in urine samples from substance use disorder clinics. Ten-microliter urine samples were deposited directly on paper spray cartridges previously loaded with internal standards, dried, and analyzed with no other sample treatment. Quantitative results were obtained using MS/MS. Individual drugs were identified using high-resolution accurate mass spectrometry, and confirmed by data-dependent MS/MS. RESULTS: Calibration curves in urine were linear over a range of 0.5-50 ng/mL with R2 of 0.99 or better for eight representative fentanyl analogs. Cartridges preloaded with internal standards demonstrated satisfactory quantitative results compared with LC/MS. Direct identification and confirmation of fentanyl analogs and other common drugs of abuse in urine using high-resolution accurate mass and MS/MS fragmentation were demonstrated at low picogram levels. CONCLUSIONS: Paper spray mass spectrometry can reliably identify and quantitate fentanyl analogs and other drugs of abuse in urine. Using paper spray cartridges as collection devices reduces exposure and transportation risks associated with biological fluids. Cartridges preloaded with labeled internal standards can be effective for targeted screening of fentanyl analogs and other drugs of abuse.

Mass spectrometry imaging of levofloxacin distribution in TB-infected pulmonary lesions by MALDI-MSI and continuous liquid microjunction surface sampling.

A multi-modal mass spectrometry imaging (MSI) and profiling approach has been applied to assess the partitioning of the anti-TB fluoroquinolone levofloxacin into pulmonary lesions. Matrix-assisted laser desorption ionization mass spectrometry imaging (MALDI-MSI) and a commercial liquid microjunction surface sampling technology (LMJ-SSP), or flowprobe, have been used to both spatially profile and image drug distributions in lung tissue sections from TB-infected rabbits following oral administration of a single human-equivalent dose. Levofloxacin levels were highest at 6 h post-dose in normal lung, cellular granuloma, and necrotic caseum compartments. The drug accumulated in the cellular granuloma regions with lower amounts partitioning into central caseous compartments. Flowprobe imaging at 630 µm (limited by the probe tip diameter) enabled visualization of drug distribution into lesion compartments, including limited differentiation of relative drug abundance in cellular versus caseous regions of the lesions. MALDI-MSI analysis at 75 µm provided more detailed drug distribution, which clearly accumulated in the cellular region immediately surrounding the central caseum core. Imaging and profiling data acquired by flowprobe and MALDI-MSI were validated by quantitative LC/MS/MS analysis of lung and granuloma homogenates taken from the same animals. The results of the investigation show flowprobe imaging and sampling as a rapid and sensitive alternative to MALDI-MSI for profiling drug distributions into tissues when spatial resolution of data below the threshold of the probe diameter is not required.

Analysis of dried blood spots using DESI mass spectrometry.

Dried blood spot (DBS) analysis using mass spectrometry is an invaluable technique for examining blood markers of inborn metabolic diseases in clinical laboratories. Implementation of DBS sampling and analysis in pharmaceutical development have more recently gained traction due to the advantages of convenience in sample procurement and logistics, as well as the innate advantages associated with the collection of lower blood volumes. While there are several realized advantages of DBS, the bioanalytical laboratory is disadvantaged and burdened with additional preparative steps prior to analysis. Therefore, improvements in the laboratory workflow for DBS analysis are necessary. Here, we describe direct blood spot analysis using desorption electrospray ionization (DESI) mass spectrometry for quantitative determination of drugs in whole blood.

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.

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.

Elution, partial separation, and identification of lipids directly from tissue slices on planar chromatography media by desorption electrospray ionization mass spectrometry.

A method for the examination of intact tissue sections for gangliosides and other lipids using desorption electrospray ionization (DESI) mass spectrometry (MS) is presented. In the present work, thin tissue slices (16 µm) taken from the rat brain are thaw mounted onto planar chromatographic media and the lipids are eluted, partially separated, and analyzed directly from the plate by DESI-MS in the negative ion mode. With the lanes scanned parallel to the direction of the chromatographic separation in the full scan mode, the selected ion current associated with ions of separated lipid molecules is plotted in order of increasing Rf values. Distinctly different classes of lipids are detected using this method, including several ganglioside species (i.e., GQ1, GT1, GD1, and GM1) and sulfoglycosphingolipids. For the examination of gangliosides in the full scan negative ion mode from high-performance thin-layer chromatography (HPTLC) plates, the limit of detection (LOD) was determined to be approximately 3 pmol. Tandem mass spectrometry (MS/MS) using the linear ion trap was used to confirm the presence of selected gangliosides and other lipids directly from the HPTLC plate. DESI-MS/MS revealed the presence of both the GD1a and GD1b isomers. The simplicity of this approach where planar separations are relied upon for sample preparation and presentation to the MS should allow for the examination of a variety of complex samples including the rapid examination of foodstuffs, bacteria, whole blood, and needle biopsies for cancer diagnostics.

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.

Desorption electrospray ionization-mass spectrometry for the detection of analytes extracted by thin-film molecularly imprinted polymers.

Desorption electrospray ionization-mass spectrometry (DESI-MS) is a powerful technique for the analysis of solid and liquid surfaces that has found numerous applications in the few years since its invention. For the first time, it is applied to the detection of analytes extracted by molecularly imprinted polymers (MIPs) in a thin-film format. MIPs formed with 2,4-dichlorophenoxyacetic acid (2,4-D) as the template were used for the extraction of this analyte from aqueous solutions spiked at concentrations of 0.0050-2.0 mg L(-1) (approximately 2 x 10(-8) to approximately 1 x 10(-5) M). The response was linear up to 0.50 mg L(-1), and then levelled off due to saturation of the active sites of the MIP. In MS/MS mode, the signal at 0.0050 mg L(-1) was still an order of magnitude higher than the signal of a blank. The MIP DESI-MS approach was also used for the analysis of tap water and river water spiked with 2,4-D and four analogues, which indicated that these analogues were also extracted to various extents. For practical applications of the MIP, a detection technique is required that can distinguish between these structurally similar compounds, and DESI-MS fulfills this purpose.

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.

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.

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.

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 microg/g (brain) to a high of 10.6 microg/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.

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 microm resolution typically takes between 30 min and 2 h.

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.

Desorption Electrospray Ionization (DESI) Analysis of Intact Proteins/Oligopeptides.

INTRODUCTIONDesorption electrospray ionization (DESI) is amenable to the study of intact proteins in complex mixtures, including blood or other biological media. Intact proteins can be desorbed and ionized from the surface under gentle (soft) conditions to produce compact conformations of the protein. A procedure for DESI analysis of intact proteins and oligopeptides using mass spectrometry (MS) is described here. DESI-MS is an emerging technique with great promise, but its application range is still being investigated. Therefore, the protocol presented here provides general procedures used for the applications that have been investigated so far. Optimal ion source parameters and surface types may vary depending on the application.

Desorption Electrospray Ionization (DESI) Analysis of Tryptic Digests/Peptides.

INTRODUCTIONThe analytical utility of desorption electrospray ionization (DESI) is such that it can be applied to qualitative proteomics research in the same way as matrix-assisted laser desorption/ionization (MALDI) and electrospray ionization (ESI) methods, although little work has yet been reported in this regard. Because DESI is a surface analysis technique and easily automated, it can be implemented for high-throughput applications, which include the analysis of chromatographic fractions of digested proteins. The analysis of tryptic peptides follows the same protocols as in typical MALDI or ESI methods, except that the mixture is spotted directly onto an insulating surface, allowed to dry, and analyzed directly without adding matrix compounds (as in the case of MALDI methods). The spectral characteristics are similar to those of ESI in that both singly and multiply charged analyte ions are detected. Spectra are highly similar to electrospray spectra of tryptic digests with regard to the overwhelming presence of multiply charged ions of peptides. DESI-mass spectrometry (DESI-MS) is an emerging technique with great promise, but its application range is still being investigated. Therefore, the protocol for DESI-MS analysis of tryptic digests/peptides presented here provides general procedures used for the applications that have been investigated so far. Optimal ion source parameters and surface types may vary, depending on the application.

In Situ Desorption Electrospray Ionization (DESI) Analysis of Tissue Sections.

INTRODUCTIONDesorption electrospray ionization (DESI) allows in situ analysis of biological tissues. The analysis of less abundant protein constituents within a tissue sample often requires the removal of lipid species prior to analysis, similar to the situation with matrix-assisted laser desorption/ionization (MALDI). After removal of lipid constituents, the tissue can be treated with protease to degrade proteins present in the tissue. The tryptic products can be investigated directly from the tissue using DESI. The spectra obtained feature ions of tryptic fragments from abundant proteins present in the tissue sample. The digestion is usually not complete; hence, the presence of missed cleavage sites is typical in the peptides detected. The signal is more stable for longer times than in the case of deposited samples, so the recording of mass spectrometry (MS)/MS data is simple in this case. DESI-MS is an emerging technique with great promise, but its application range is still being investigated. Therefore, the protocol for DESI-MS analysis of tissue sections presented here provides general procedures used for the applications that have been investigated so far. Optimal ion source parameters and surface types may vary depending on the application.

Desorption Electrospray Ionization: Proteomics Studies by a Method That Bridges ESI and MALDI.

INTRODUCTIONDesorption electrospray ionization (DESI) is a desorption ionization (DI) method by nature, and, like matrix-assisted laser desorption/ionization (MALDI), it is used for the analysis of material present on a surface. DESI includes features reminiscent of electrospray ionization (ESI) in respect to both its instrumental and mechanistic aspects. However, the analyte in the DESI experiment is not in solution as in ESI. Instead, a microelectrospray ion source is used to produce charged droplets, ionic clusters, and/or gas-phase ions (depending on chosen experimental conditions), and these are directed at the sample surface. The sample is present in the ambient environment. An electrical potential of several kilovolts (kV) is applied to the spray solution, and pneumatic nebulization is used to assist in desolvation. Ionization of molecules present on the sample surface occurs upon the impact of the ESI-originated, charged particles with the surface. Surfaces include deposited samples on sample holder targets as well as surfaces of natural objects such as biological tissues or minerals.