Multiplex Mass Spectrometric Imaging with Polarity Switching for Concurrent Acquisition of Positive and Negative Ion Images

We have recently developed a multiplex mass spectrometry imaging (MSI) method which incorporates high mass resolution imaging and MS/MS and MS³ imaging of several compounds in a single data acquisition utilizing a hybrid linear ion trap-Orbitrap mass spectrometer (Perdian and Lee, Anal. Chem. 82, 9393–9400, 2010). Here we extend this capability to obtain positive and negative ion MS and MS/MS spectra in a single MS imaging experiment through polarity switching within spiral steps of each raster step. This methodology was demonstrated for the analysis of various lipid class compounds in a section of mouse brain. This allows for simultaneous imaging of compounds that are readily ionized in positive mode (e.g., phosphatidylcholines and sphingomyelins) and those that are readily ionized in negative mode (e.g., sulfatides, phosphatidylinositols and phosphatidylserines). MS/MS imaging was also performed for a few compounds in both positive and negative ion mode within the same experimental set-up. Insufficient stabilization time for the Orbitrap high voltage leads to slight deviations in observed masses, but these deviations are systematic and were easily corrected with a two-point calibration to background ions.

Blotting Assisted by Heating and Solvent Extraction for DESI-MS Imaging

Imprints of potato sprout (Solanum tuberosum L.), gingko leaves (Gingko biloba L.) and strawberries (Fragaria x ananassa Duch.) were successfully imaged by desorption electrospray ionization mass spectrometry (DESI-MS) on TLC plates through blotting assisted by heating and/or solvent extraction. Ion images showing the distribution of significant compounds such as glycoalkaloid toxins in potato sprout, ginkgolic acids and flavonoids in ginkgo leaves, and sugars and anthocyanidin in strawberry were obtained. Practical implications of this work include analysis of a wide range of irregular or soft materials by different imprinting conditions without requiring the addition of matrices or use of specific kinds of surfaces.

Distribution study of atorvastatin and its metabolites in rat tissues using combined information from UHPLC/MS and MALDI-Orbitrap-MS imaging

The combination of ultrahigh-resolution mass spectrometry imaging (UHRMSI) and ultrahigh-performance liquid chromatography coupled with tandem mass spectrometry (UHPLC/MS/MS) was used for the identification and the spatial localization of atorvastatin (AT) and its metabolites in rat tissues. Ultrahigh-resolution and high mass accuracy measurements on a matrix-assisted laser desorption/ionization (MALDI)-Orbitrap mass spectrometer allowed better detection of desired analytes in the background of matrix and endogenous compounds. Tandem mass spectra were also used to confirm the identification of detected metabolites in complex matrices. The optimization of sample preparation before imaging experiments included the tissue cryogenic sectioning (thickness 20 μm), the transfer to stainless steel or glass slide, and the selection of suitable matrix and its homogenous deposition on the tissue slice. Thirteen matrices typically used for small molecule analysis, e.g., 2,5-dihydroxybenzoic acid (DHB), 1,5-diaminonaphthalene (DAN), 9-aminoacridine (AA), etc., were investigated for the studied drug and its metabolite detection efficiency in both polarity modes. Particular matrices were scored based on the strength of extracted ion current (EIC), relative ratio of AT molecular adducts, and fragment ions. The matrix deposition on the tissue for the most suitable matrices was done by sublimation to obtain the small crystal size and to avoid local variations in the ionization efficiency. UHPLC/MS profiling of drug metabolites in adjacent tissue slices with the previously optimized extraction was performed in parallel to mass spectrometry imaging (MSI) measurements to obtain more detailed information on metabolites in addition to the spatial information from MSI. The quantitation of atorvastatin in rat liver, serum, and feces was also performed.

Improved MALDI-TOF Microbial Mass Spectrometry Imaging by Application of a Dispersed Solid Matrix

The key step in high quality microbial matrix-assisted laser desorption/ionization mass spectrometry imaging (microbial MALDI MSI) is the fabrication of a homogeneous matrix coating showing a fine-grained morphology. This application note addresses a novel method to apply solid MALDI matrices onto microbial cultures grown on thin agar media. A suspension of a mixture of 2,5-DHB and α-CHCA is sprayed onto the agar sample surface to form highly homogeneous matrix coatings. As a result, the signal intensities of metabolites secreted by the fungus Aspergillus fumigatus were found to be clearly enhanced.

Three-dimensional molecular reconstruction of rat heart with mass spectrometry imaging

Cardiovascular diseases are the world’s number one cause of death, accounting for 17.1 million deaths a year. New high-resolution molecular and structural imaging strategies are needed to understand underlying pathophysiological mechanism. The aim of our study is (1) to provide a molecular basis of the heart animal model through the local identification of biomolecules by mass spectrometry imaging (MSI) (three-dimensional (3D) molecular reconstruction), (2) to perform a cross-species validation of secondary ion mass spectrometry (SIMS)-based cardiovascular molecular imaging, and (3) to demonstrate potential clinical relevance by the application of this innovative methodology to human heart specimens. We investigated a MSI approach using SIMS on the major areas of a rat and mouse heart: the pericardium, the myocardium, the endocardium, valves, and the great vessels. While several structures of the heart can be observed in individual two-dimensional sections analyzed by metal-assisted SIMS imaging, a full view of these structures in the total heart volume can be achieved only through the construction of the 3D heart model. The images of 3D reconstruction of the rat heart show a highly complementary localization between Na⁺, K⁺, and two ions at m/z 145 and 667. Principal component analysis of the MSI data clearly identified different morphology of the heart by their distinct correlated molecular signatures. The results reported here represent the first 3D molecular reconstruction of rat heart by SIMS imaging.

Negative ion tandem mass spectrometry of prenylated fungal metabolites and their derivatives

Liquid chromatography negative ion electrospray ionisation tandem mass spectrometry has been used for characterisation of naturally occurring prenylated fungal metabolites and synthetic derivatives. The fragmentation studies allow an elucidation of the decomposition pathways for these compounds. It could be shown, that the prenyl side chain is degraded by successive radical losses of C₅ units. Both the benzoquinones and the phenolic derivatives display significant key ions comprising the aromatic ring. In some cases, the formation of significant oxygen-free key ions could be evidenced by high-resolution MS/MS measurements. Furthermore, the different types of basic skeletons, benzoquinones and phenol type as well as cyclic prenylated compounds, can be differentiated by their MS/MS behaviour.

Quantitative Surface Analysis of a Binary Drug Mixture—Suppression Effects in the Detection of Sputtered Ions and Post-Ionized Neutrals

A systematic mass spectrometric study of two of the most common analgesic drugs, paracetamol and ibuprofen, is reported. The drugs were studied by means of secondary ion mass spectrometry (SIMS) and secondary neutral mass spectrometry (SNMS) using laser post-ionization (LPI) both in pure samples and in a two-component mixture. Ion suppression within the two-component system observed in SIMS mode is ameliorated using LPI under room temperature analysis. However, suppression effects are apparent in LPI mode on performing the analysis at cryogenic temperatures, which we attribute to changes in the desorption characteristics of sputtered molecules, which influences the subsequent post-ionization efficiency. This suggests different mechanisms of ion suppression in SIMS and LPI modes.

Conventional liquid chromatography/triple quadrupole mass spectrometry based metabolite identification and semi-quantitative estimation approach in the investigation of in vitro dabigatran etexilate metabolism

Dabigatran etexilate (DABE) is an oral prodrug that is rapidly converted by esterases to dabigatran (DAB), a direct inhibitor of thrombin. To elucidate the esterase-mediated metabolic pathway of DABE, a high-performance liquid chromatography/mass spectrometry based metabolite identification and semi-quantitative estimation approach was developed. To overcome the poor full-scan sensitivity of conventional triple quadrupole mass spectrometry, precursor–product ion pairs were predicted to search for the potential in vitro metabolites. The detected metabolites were confirmed by the product ion scan. A dilution method was introduced to evaluate the matrix effects on tentatively identified metabolites without chemical standards. Quantitative information on detected metabolites was obtained using “metabolite standards” generated from incubation samples that contain a high concentration of metabolite in combination with a correction factor for mass spectrometry response. Two in vitro metabolites of DABE (M1 and M2) were identified, and quantified by the semi-quantitative estimation approach. It is noteworthy that CES1 converts DABE to M1 while CES2 mediates the conversion of DABE to M2. M1 and M2 were further metabolized to DAB by CES2 and CES1, respectively. The approach presented here provides a solution to a bioanalytical need for fast identification and semi-quantitative estimation of CES metabolites in preclinical samples.

Rapid and sensitive analysis of phthalate metabolites, bisphenol A, and endogenous steroid hormones in human urine by mixed-mode solid-phase extraction, dansylation, and ultra-performance liquid chromatography coupled with triple quadrupole mass spectrometry

Steroid hormone levels in human urine are convenient and sensitive indicators for the impact of phthalates and/or bisphenol A (BPA) exposure on the human steroid hormone endocrine system. In this study, a rapid and sensitive method for determination of 14 phthalate metabolites, BPA, and ten endogenous steroid hormones in urine was developed and validated on the basis of ultra-performance liquid chromatography coupled with electrospray ionization triple quadrupole mass spectrometry. The optimized mixed-mode solid phase-extraction separated the weakly acidic or neutral BPA and steroid hormones from acidic phthalate metabolites in urine: the former were determined in positive ion mode with a methanol/water mobile phase containing 10 mM ammonium formate; the latter were determined in negative ion mode with a acetonitrile/water mobile phase containing 0.1 % acetic acid, which significantly alleviated matrix effects for the analysis of BPA and steroid hormones. Dansylation of estrogens and BPA realized simultaneous and sensitive analysis of the endogenous steroid hormones and BPA in a single chromatographic run. The limits of detection were less than 0.84 ng/mL for phthalate metabolites and less than 0.22 ng/mL for endogenous steroid hormones and BPA. This proposed method had satisfactory precision and accuracy, and was successfully applied to the analyses of human urine samples. This method could be valuable when investigating the associations among endocrine-disrupting chemicals, endogenous steroid hormones, and relevant adverse outcomes in epidemiological studies.

High-resolution atmospheric pressure infrared laser desorption/ionization mass spectrometry imaging of biological tissue

An atmospheric pressure laser desorption/ionization mass spectrometry imaging ion source has been developed that combines high spatial resolution and high mass resolution for the in situ analysis of biological tissue. The system is based on an infrared laser system working at 2.94 to 3.10 μm wavelength, employing a Nd:YAG laser-pumped optical parametrical oscillator. A Raman-shifted Nd:YAG laser system was also tested as an alternative irradiation source. A dedicated optical setup was used to focus the laser beam, coaxially with the ion optical axis and normal to the sample surface, to a spot size of 30 μm in diameter. No additional matrix was needed for laser desorption/ionization. A cooling stage was developed to reduce evaporation of physiological cell water. Ions were formed under atmospheric pressure and transferred by an extended heated capillary into the atmospheric pressure inlet of an orbital trapping mass spectrometer. Various phospholipid compounds were detected, identified, and imaged at a pixel resolution of up to 25 μm from mouse brain tissue sections. Mass accuracies of better than 2 ppm and a mass resolution of 30,000 at m/z?=?400 were achieved for these measurements.

High-resolution MALDI-TOF mass spectrometry of bacterial proteins using a Tris-EDTA buffer approach

The performance of matrix assisted laser desorption-ionization time of flight mass spectrometry (MALDI-TOF) of bacterial proteins strongly depends on sample preparation. It is found that the mass spectral profiles obtained from direct MALDI-TOF MS of the protein extracts are much weaker for individual bacterial cells than compared to those prepared by the Tris-EDTA buffer approach (TEBA). Characteristic mass spectral peaks were observed in the mass range from 3,000 to 15,000?Da. The mass peaks reported earlier and claimed to serve as species-specific biomarkers are consistently found here as well. Mass peaks at m/z of 3636, 5466, 5750, 6315, 6547, 7274, 9192, and 9742 are found for Escherichia coli studied and assigned as specific biomarkers. Similarly, specific mass peaks have been identified at m/z 5443, 7270, 7724, and 9888 for Bacillus subtilis, and at 3603, 5496, 6800, 8858 and 9531 for Serratia marcescens. The detection limits for the three target bacteria range from 2.4?×?10⁵ to 3.3?×?10⁵?cfu·mL^-1. We conclude that the TE buffer approach can produce reliable data for rapid classification, high-resolution and highly sensitive detection of bacteria.

MALDI-FTICR MS Imaging as a Powerful Tool to Identify Paenibacillus Antibiotics Involved in the Inhibition of Plant Pathogens

Nowadays, microorganisms are more and more often used as biocontrol agents for crop protection against diseases. Among them, bacteria of Bacillus and Paenibacillus genders are already used as commercial biocontrol agents. Their mode of action is supposed to be related to their production of antibiotics, such as cyclic lipopeptides, which exhibit great antimicrobial activities. We chose to work with a Paenibacillus polymyxa strain (Pp56) very resistant to various microorganisms. The bacteria were grown simultaneously with Fusarium oxysporum and we applied matrix-assisted laser desorption/ionization-Fourier transform ion cyclotron resonance (MALDI-FTICR) mass spectrometry to identify the antibiotics compounds present in the fungus growth inhibition area. We, therefore, identified fusaricidins A, B, and C and numerous members of the LI-F antibiotics family. MALDI-FTICR mass spectrometry imaging was then used to follow the diffusion of lipopeptides involved in the inhibitory activity over time. We analyzed the molecular content of the inhibitory area at different Pp56 and Fusarium incubation durations and concluded that some lipopeptides such as fusaricidin B and a mixture of LI-F05b/06b/08a were mainly involved in the defense mechanism of Pp56. Our study confirms that MALDI imaging may be a powerful tool to quickly determine which molecular species is involved in an antagonism with another microorganism, avoiding time-consuming steps of extraction, purification, and activity tests, which are still commonly used in microbiology.

Determination of benzyl isothiocyanate metabolites in human plasma and urine by LC-ESI-MS/MS after ingestion of nasturtium (Tropaeolum majus L.)

A liquid chromatography–tandem mass spectrometry (LC-MS/MS) method was developed and validated to determine the concentration of benzyl isothiocyanate (BITC) metabolites in human plasma and urine. In this study, the following BITC metabolites have been considered: BITC–glutathione, BITC–cysteinylglycine, BITC–cysteine, and BITC–N-acetyl-l-cysteine. The assay development included: (1) synthesis of BITC conjugates acting as reference substances; (2) sample preparation based on protein precipitation and solid-phase extraction; (3) development of a quantitative LC-MS/MS method working in the multiple-reaction monitoring mode; (4) validation of the assay; (5) investigation of the stability and the reactivity of BITC conjugates in vitro; (6) application of the method to samples from a human intervention study. The lower limits of quantification were in the range of 21–183 nM depending on analyte and matrix, whereas the average recovery rates from spiked plasma and urine were approximately 85 and 75 %, respectively. BITC conjugates were found to be not stable in alkaline buffered solutions. After consumption of nasturtium, containing 1,000 μM glucotropaeolin, the primary source of BITC, quantifiable levels of BITC–NAC, BITC–Cys, and BITC–CysGly were found in human urine samples. Maximum levels in urine were determined 4 h after the ingestion of nasturtium. With regard to the human plasma samples, all metabolites were determined including individual distributions. The work presented provides a validated LC-MS/MS method for the determination of BITC metabolites and its successful application for the analysis of samples collected in a human intervention study.

Infrared Matrix-Assisted Laser Desorption Electrospray Ionization (IR-MALDESI) Imaging Source Coupled to a FT-ICR Mass Spectrometer

Mass spectrometry imaging (MSI) allows for the direct monitoring of the abundance and spatial distribution of chemical compounds over the surface of a tissue sample. This technology has opened the field of mass spectrometry to numerous innovative applications over the past 15 years. First used with SIMS and MALDI MS that operate under vacuum, interest has grown for mass spectrometry ionization sources that allow for effective imaging but where the analysis can be performed at ambient pressure with minimal or no sample preparation. We introduce here a versatile source for MALDESI imaging analysis coupled to a hybrid LTQ-FT-ICR mass spectrometer. The imaging source offers single shot or multi-shot capability per pixel with full control over the laser repetition rate and mass spectrometer scanning cycle. Scanning rates can be as fast as 1 pixel/second and a spatial resolution of 45 μm was achieved with oversampling.

Qualitative metabolism assessment and toxicological detection of xylazine, a veterinary tranquilizer and drug of abuse, in rat and human urine using GC–MS, LC–MS n , and LC–HR-MS n

Xylazine is used in veterinary medicine for sedation, anesthesia, and analgesia. It has also been reported to be misused as a horse doping agent, a drug of abuse, a drug for attempted sexual assault, and as source of accidental or intended poisonings. So far, no data concerning human metabolism have been described. Such data are necessary for the development of toxicological detection methods for monitoring drug abuse, as in most cases the metabolites are the analytical targets. Therefore, the metabolism of xylazine was investigated in rat and human urine after several sample workup procedures. The metabolites were identified using gas chromatography (GC)–mass spectrometry (MS) and liquid chromatography (LC) coupled with linear ion trap high-resolution multistage MS (MS ⁿ ). Xylazine was N-dealkylated and S-dealkylated, oxidized, and/or hydroxylated to 12 phase I metabolites. The phenolic metabolites were partly excreted as glucuronides or sulfates. All phase I and phase II metabolites identified in rat urine were also detected in human urine. In rat urine after a low dose as well as in human urine after an overdose, mainly the hydroxy metabolites were detected using the authors’ standard urine screening approaches by GC–MS and LC–MS ⁿ . Thus, it should be possible to monitor application of xylazine assuming similar toxicokinetics in humans.

Laser desorption VUV postionization MS imaging of a cocultured biofilm

Laser desorption postionization mass spectrometry (LDPI-MS) imaging is demonstrated with a 10.5 eV photon energy source for analysis and imaging of small endogenous molecules within intact biofilms. Biofilm consortia comprised of a synthetic Escherichia coli K12 coculture engineered for syntrophic metabolite exchange are grown on membranes and then used to test LDPI-MS analysis and imaging. Both E. coli strains displayed many similar peaks in LDPI-MS up to m/z 650, although some observed differences in peak intensities were consistent with the appearance of byproducts preferentially expressed by one strain. The relatively low mass resolution and accuracy of this specific LDPI-MS instrument prevented definitive assignment of species to peaks, but strategies are discussed to overcome this shortcoming. The results are also discussed in terms of desorption and ionization issues related to the use of 10.5 eV single-photon ionization, with control experiments providing additional mechanistic information. Finally, 10.5 eV LDPI-MS was able to collect ion images from intact, electrically insulating biofilms at ～100 μm spatial resolution. Spatial resolution of ～20 μm was possible, although a relatively long acquisition time resulted from the 10 Hz repetition rate of the single-photon ionization source.

Occurrence of venlafaxine residues and its metabolites in marine mussels at trace levels: development of analytical method and a monitoring program

Coastal areas are subject to growing pressures and impacts because of the increase in human activities. Lipophilic organic contaminants, such as polycyclic aromatic hydrocarbons (PAHs) or polychlorinated biphenyls (PCBs), have been monitored for decades within monitoring programs. However, until now, little information on the detection of so-called “emerging contaminants” such as hydrophilic organic compounds in the marine environment and no data on its metabolites or transformation products in marine organisms is available. In this report, a sensitive analytical methodology for identification and confirmation of venlafaxine (VEN) residues and five of its main metabolites in the marine mussels Mytilus galloprovincialis was validated. The sample preparation procedure was based on the Quick, Easy, Cheap, Effective, Rugged, and Safe (QuEChERS) approach. An analytical method was developed to quantify these compounds at trace levels by liquid chromatography coupled to high-resolution mass spectrometry. The method was then applied to marine mussels collected from the Mediterranean Sea in southeastern France. Residues of the antidepressant VEN were occasionally detected at ng/g dw level. In addition, the approach allowed us to identify several transformation products in the analyzed samples. N-desmethylvenlafaxine (NDV) was the most frequently detected metabolite followed by N,O-di-desmethylvenlafaxine (NODDV).

Toward high spatial resolution sampling and characterization of biological tissue surfaces using mass spectrometry

Mass spectrometry has emerged as a powerful tool for the bioanalytical sciences because of its ability to characterize small and large biomolecules in vanishingly small amounts. A recurring motif in mass spectrometry aims to decipher the chemical composition of biological samples at the molecular level, requiring drastic improvements in the ability to interrogate well defined and highly spatially resolved areas of a sample surface. With the growth of novel ionization methods, numerous advances have been made in sampling biological tissue surfaces. Here, current advancements in ambient, inlet, and vacuum ionization methods are discussed with respect to the potential improvements in the goal of achieving high spatial resolution and/or fast surface analysis. Of similar importance is the need for improvements in applicable characterization strategies using high performance fragmentation technologies such as electron transfer dissociation and electron capture dissociation directly from surfaces, and gas-phase separation through ion mobility spectrometry and high resolution mass spectrometry.

Overcoming Selectivity and Sensitivity Issues of Direct Inject Electrospray Mass Spectrometry via DAPNe-NSI-MS

Direct inject electrospray mass spectrometry offers minimal sample preparation and a “shotgun” approach to analyzing samples. However, complex matrix effects often make direct inject an undesirable sample introduction technique, particularly for trace level analytes. Highlighted here is our solution to the pitfalls of direct inject mass spectrometry and other ambient ionization methods with a focus on trace explosives. Direct analyte-probed nanoextraction coupled to nanospray ionization mass spectrometry solves selectivity issues and reduces matrix effects while maintaining minimal sample preparation requirements. With appropriate solvent conditions, most explosive residues can be analyzed with this technique regardless of the nature of the substance (i.e., nitroaromatic, oxidizing salt, or peroxide).

Rapid screening of active ingredients in drugs by mass spectrometry with low-temperature plasma probe

A high-throughput method for rapid screening of active ingredients in drugs has been developed with mass spectrometry coupled to a low-temperature plasma (LTP) probe ion source. Without sample preparation or pretreatment, the active ingredients of 11 types of commercial pharmaceuticals, including hormones, antipyretic analgesics, cardiovascular, digestant, neuro-psychotherapeutic, diuretic, antithyroid, sulfa anti-inflammatory, antiparastic, sedative-hypnotics, and antibacterial, were directly desorbed/ionized and detected by a linear ion trap mass spectrometry (MS). The structures of these ingredients were elucidated by tandem MS. The analysis of 18 methyltestosterone tablets could be accomplished within 1.9 min, which allows fast detection with a speed of approximate 600 samples within 1 h. This work demonstrated that LTP probe ion source combined with MS is a high-throughput method for screening of pharmaceuticals and potentially applied to on-line quality control in pharmaceutical industry.