Ion mobility-mass spectrometry (IM-MS) has recently seen increased use in the analysis of small molecules, especially in the field of metabolomics, for increased breadth of information and improved separation of isomers. In this study, steroid epimers androsterone and trans-androsterone were analyzed with IM-MS to investigate differences in their relative mobilities. Although sodiated monomers exhibited very similar collision cross-sections (CCS), baseline separation was observed for the sodiated dimer species (RS = 1.81), with measured CCS of 242.6 and 256.3 Å2, respectively. Theoretical modeling was performed to determine the most energetically stable structures of solution-phase and gas-phase monomer and dimer structures. It was revealed that these epimers differ in their preferred dimer binding mode in solution phase: androsterone adopts a R=O – Na+ – OH—R′ configuration, whereas trans-androsterone adopts a R=O – Na+ – O=R′ configuration. This difference contributes to a significant structural variation, and subsequent CCS calculations based on these structures relaxed in the gas phase were in agreement with experimentally measured values (ΔCCS ~ 5%). Additionally, these calculations accurately predicted the relative difference in mobility between the epimers. This study illustrates the power of combining experimental and theoretical results to better elucidate gas-phase structures.

Drift tube ion mobility spectrometry (DTIMS) coupled with mass spectrometry was evaluated for its capabilities in rapid separation of endogenous isomeric steroids. These compounds, which included eight isomer groups, were investigated as protonated and sodiated species and collision cross sections were measured for all ionization species of each steroid. Pregnenolone (CCSN2 176.7 Å2) and 5α-dihydroprogesterone (CCSN2 191.4 Å2) could be separated as protonated species, and aldosterone (CCSN2 197.7 Å2) and cortisone (CCSN2 211.7 Å2) could be separated as sodiated monomers. However, the sodiated dimers of the remaining isomers yielded increased separation, resulting in baseline resolution. Specific structural differences including ring conformation and the chirality of hydroxyl groups were compared to evaluate their relative effects on collision cross section in isomers. These results indicated that C5 ring conformation isomers androsterone and etiocholanolone, which both contain a C3 α-hydroxyl group, yielded similar dimer CCS. Yet these compounds were well resolved from their respective β-hydroxyl epimers, trans-androsterone and epietiocholanolone. Alternative drift gases were evaluated, and carbon dioxide drift gas offered slight improvement in isomer resolution well, including allowing separation of testosterone (CCSCO2 330.0 Å2), dehydroepiandrosterone (CCSCO2 312.6 Å2), and epitestosterone (CCSCO2 305.6 Å2). Finally, different metal cation adducts, including alkali, alkaline earth, and first row transition metal adducts were analyzed, and several of these species provided improved resolution between steroid epimers. Overall, this study shows that drift tube ion mobility is a promising tool for improved separation of isomeric steroids.

•LDI and MALDI cause reduction of anthraquinones, which is observed as [M + 2H]+ ions.•The reduction site of anthraquinones is the carbonyl, which was elucidated by MS/MS.•Anthraquinone undergoes UV photoreduction, which was confirmed by HPLC/UV/ESI‐MS/MS.•Reduction extent of alizarin and anthraquinone increases with concentration.•Alizarin was detected in its reduced form in a painting cross section and dyed fabric.The peculiar ionization of anthraquinones, including the artistic dyes alizarin and purpurin, is examined. When alizarin (MW = 240) is ionized by either LDI or MALDI it exhibits a dominant ion of m/z 242 [M + 2H]+ with a far greater abundance than expected from the 13C isotopic contribution from the [M + H]+ ion at m/z 241. For the first time, MS/MS analysis of these anomalous [M + 2H]+ ions is presented, which indicates that they arise from a laser-induced photoreduction of one of the anthraquinone’s carbonyl groups. MS/MS daughter ions produced from neutral losses of either water or ammonia from the [M + 2H]+ ions exhibit different relative abundances, depending upon their respective functional groups’ proximity to the reduced carbonyl. LDI-MS/MS was used for the in situ detection of alizarin in a painting cross section and a swatch of dyed silk.Download high-res image (91KB)Download full-size image

•Solvent vapor added to planar FAIMS cell dramatically increases resolving power.•Magnitude of the solvent effect correlates with the solvent molecular volume.•Consistent with clustering/declustering of ions with neutral molecules.•Heat lessens the solvent vapor effect, consistent with cluster/decluster model.Solvent vapor added to the drift gas in a planar high-field asymmetric waveform ion mobility spectrometer (FAIMS) leads to dramatic shifts to larger CV values while retaining narrow peaks, leading to dramatically increased resolving power (up to ∼140) for a variety of ions including isomeric ions. Here we present studies designed to improve the fundamental understanding of the role of solvent vapor on performance of planar FAIMS, focusing on solvent trends with molecular size and effects of temperature. The studies employ as test compounds the [M − H]− ions of the three positional isomers of phthalic acid and the M− and [M − H]− ions of trinitrotoluene (TNT). The addition of solvent vapor to FAIMS carrier gas has a significant impact on the behavior of the ions inside the FAIMS cell and can impact the overall performance of the cell. We have shown that by comparing a range of solvent vapors (water and C1–C4 alcohols), the magnitude of the effect correlates to the molecular volume of the solvent molecule. This fits well with the model of clustering/declustering of ions with neutral molecules on the time scale of waveform used in FAIMS. We have also shown that with the addition of heat, the effect of the solvent vapor is lessened, as one would expect with the cluster/decluster model.

Skeletal muscles are composed of heterogeneous muscle fibers that have different physiological, morphological, biochemical, and histological characteristics. In this work, skeletal muscles extensor digitorum longus, soleus, and whole gastrocnemius were analyzed by matrix-assisted laser desorption/ionization mass spectrometry to characterize small molecule metabolites of oxidative and glycolytic muscle fiber types as well as to visualize biomarker localization. Multivariate data analysis such as principal component analysis (PCA) and partial least squares discriminant analysis (PLS-DA) were performed to extract significant features. Different metabolic fingerprints were observed from oxidative and glycolytic fibers. Higher abundances of biomolecules such as antioxidant anserine as well as acylcarnitines were observed in the glycolytic fibers, whereas taurine and some nucleotides were found to be localized in the oxidative fibers.

•Folch, Matyash, and Ostro plate preparations were compared for lipid coverage.•Matyash method provides recovery and reproducibility across lipid classes.•The Waters Ostro plate is beneficial for targeted lipidomics by class.•Matyash provides recovery of polar compounds as use in biphasic extraction.The goal of this research was to find the most comprehensive lipid extraction of blood plasma, while also providing adequate aqueous preparation for metabolite analysis. Comparisons have been made previously of the Folch, Bligh–Dyer, and Matyash lipid extractions; furthermore, this paper provides an additional comparison of a phospholipid removal plate for analysis. This plate was used for lipid extraction rather than its intended use in lipid removal for polar analysis, and it proves to be robust for targeted lipid analysis. Folch and Matyash provided reproducible recovery over a range of lipid classes, however the Matyash aqueous layer compared well to a typical methanol preparation for polar metabolite analysis. Thus, the Matyash method is the best choice for an untargeted biphasic extraction for metabolomics and lipidomics in blood plasma.

Our long-time association with Jim Morrison and the work that came from it is the result of a series of fortunate coincidences. We are pleased to be able to share recollections here of our interactions with Jim and how his life and work have influenced us and the field of mass spectrometry.

Imaging applied toward lignocellulosic materials requires high molecular specificity to map specific compounds within intact tissue. Although secondary ionization mass spectrometry (SIMS) and matrix-assisted laser desorption/ionization-mass spectrometry (MALDI-MS) with a single stage of MS have been used to image lignocellulosic biomass, the complexity of the plant tissue requires tandem MS, which limits the interpretation of simple MS. MALDI linear ion trap (LIT) tandem MS offers the high molecular specificity needed for lignocellulosic analyses. MALDI-LIT MS analyses of cellulose and xylan (hemicellulose) standards were performed to determine mass-to-charge ratios and fragmentation pathways for identification of these compounds in intact tissue. The MALDI-LIT-MS images of young Populus wood stem showed even distribution of both cellulose and hemicellulose ions; in contrast, the tandem MS images of cellulose and hemicellulose generated by plotting characteristic fragment ions resulted in drastically different images. This demonstrates that isobaric ions are present during MALDI-LIT-MS analyses of wood tissue and tandem MS is necessary to distinguish between isobaric species for selective imaging of carbohydrates in biomass.

Matrix-assisted laser desorption/ionization imaging mass spectrometry was used to analyze donor eye tissue specimens for phospholipid content to evaluate lipid distribution. Phosphatidylcholines and sphingomyelins were detected in the positive ion mode using 2,5-dihydroxybenzoic acid as the matrix. During this study, unknown ion signals in the lower m/z region (less than m/z 400) were detected, mainly in the far periphery of human flat-mounted tissue but not in age-matched rhesus monkey tissue prepared in a similar manner. The unknown ion signals occurred at m/z 304, 332, 360, and 388. These ions were subjected to tandem mass spectrometry directly from the tissue sample, and exact mass measurements of extracts were prepared for further identification. These ions were identified as alkyl dimethylbenzylammonium surfactants (benzalkonium chlorides (BACs)). The classification of these species was verified by comparing an eye tissue extract to an over-the-counter eye-care product containing BACs.

High-field asymmetric-waveform ion mobility spectrometry (FAIMS) is a rapidly growing gas-phase separation technique with a wide variety of applications. The geometry of the FAIMS cell influences ion transmission and resolution. Commercial FAIMS systems employ planar or cylindrical cell geometries. A spherical or hemispherical FAIMS cell would allow ions to travel equal path lengths through the FAIMS device while minimizing diffusional losses, improving ion resolution without sacrificing ion transmission. Here we detail the development of a novel hemispherical FAIMS cell and demonstrate the separation of a mixture of trinitrotoluene (TNT) and 3,4-dinitrotoluene (3,4-DNT). The resolution between TNT and 3,4-DNT using the hemispherical FAIMS cell is improved over that achieved with a commercial cylindrical cell while maintaining equivalent ion transmission. The novel hemispherical FAIMS cell provides improved resolution and resolving powers when compared to a commercially available cell.A novel hemispherical FAIMS cell is introduced which allows ions to travel equal path lengths while minimizing diffusional losses and improving ion resolution without sacrificing ion transmission.

Detection of drugs in tissue typically requires extensive sample preparation in which the tissue is first homogenized, followed by drug extraction, before the extracts are finally analyzed by LC/MS. Directly analyzing drugs in intact tissue would eliminate any complications introduced by sample pretreatment. A matrix-assisted laser desorption/ionization tandem mass spectrometry (MALDI-MSn) method as been developed for the quantification of cocaine present in postmortem brain tissue of a chronic human cocaine user. It is shown that tandem mass spectrometry (MS2 and MS3) increase selectivity, which is critical for differentiating analyte ions from background ions such as matrix clusters and endogenous compounds found in brain tissue. It is also shown that the use of internal standards corrects for signal variability during quantitative MALDI, which can be caused by inhomogeneous crystal formation, inconsistent sample preparation, and laser shot-to-shot variability. The MALDI-MSn method developed allows for a single MS3 experiment that uses a wide isolation window to isolate both analyte and internal standard target ions. This method is shown to provide improved precision [∼10–20 times reduction in percent relative standard deviation (%RSD)] for quantitative analysis compared to using two alternating MS3 experiments that separately isolate the target analyte and internal standard ions.

Steroid derivatization was investigated by varying the experimental parameters (reagent, reaction time, and reaction temperature) to determine the optimal conditions for individual steroids, and for larger subsets. Three methods of derivatization enhancement were also investigated: the use of sonication, the use of a microwave heating, and the addition of solvents to the reaction mixture. On a comprehensive level, derivatization using N-methyl-N-trimethylsilyl-trifluoroacetamide (MSTFA) was most efficient, while the application of solvent addition and microwave heating, in several cases, provided a clear enhancement. In addition, generalized rules for steroid derivatization are described.

The efficiency of the collision-induced dissociation (CID) process as a function of the internal energy deposited into the ion during the ionization event was evaluated. (M + H)+ ions of pyrrole, pyrrolidine, pyridine and piperidine (five and six-membered ring heterocyclics) were generated by chemical ionization (CI). The internal energy of the ions was varied by using different reagent gases. Both high-energy (keV) and low-energy (eV) CID were performed on these ions. The experiments showed that the (M + H)+ ions of the five-membered ring compounds, pyrrole and pyrrolidine, have higher fragmentation efficiencies than the six-membered ring compounds, pyridine and piperidine. Fragmentation efficiencies in high-energy CID clearly correlate with the internal energy deposited by the ionization technique. Experiments showed that the low-energy CID process is more sensitive than high-energy CID to changes in internal energy.

•The evidence on association of DEHP with adverse pregnancy outcomes is inconsistent.•Heterogeneity of the included studies precludes a direct comparison of the findings.•Some of the findings should be interpreted with caution due to design limitations.•Future studies should address methodological concerns identified in this review.Adverse pregnancy outcomes, including preterm delivery, short gestational age, and abnormal birth weight, remain a public health concern. The evidence on the association of the most common phthalate, di-2-ethylhexyl phthalate (DEHP) with adverse pregnancy outcomes remains equivocal. This systematic review summarizes published studies that investigated the association of DEHP with preterm delivery, gestational age, and birthweight. A comprehensive literature search found 15 relevant studies, most of which evaluated more than one outcome (four studies for preterm delivery, nine studies for gestational age, and ten studies for birthweight). Studies varied greatly with respect to study design, exposure assessment, analytical methods, and direction of the associations. We identified important methodological concerns which could have resulted in selection bias and exposure misclassification and contributed to null findings and biased associations. Given limitations of the previous studies discussed in this review, more thorough investigation of these associations is warranted to advance our scientific knowledge.

Detection of drugs in tissue typically requires extensive sample preparation in which the tissue is first homogenized, followed by drug extraction, before the extracts are finally analyzed by LC/MS. Directly analyzing drugs in intact tissue would eliminate any complications introduced by sample pretreatment. A matrix-assisted laser desorption/ionization tandem mass spectrometry (MALDI-MSn) method as been developed for the quantification of cocaine present in postmortem brain tissue of a chronic human cocaine user. It is shown that tandem mass spectrometry (MS2 and MS3 increase selectivity, which is critical for differentiating analyte ions from background ions such as matrix clusters and endogenous compounds found in brain tissue. It is also shown that the use of internal standards corrects for signal variability during quantitative MALDI, which can be caused by inhomogeneous crystal formation, inconsistent sample preparation, and laser shot-to-shot variability. The MALDI-MSn method developed allows for a single MS3 experiment that uses a wide isolation window to isolate both analyte and internal standard target ions. This method is shown to provide improved precision [∼10–20 times reduction in percent relative standard deviation (%RSD)] for quantitative analysis compared to using two alternating MS3 experiments that separately isolate the target analyte and internal standard ions.Isolating cocaine and cocaine-d3 in a single window improves signal reproducibility of MALDI MS/MS and MS3 for quantification of cocaine in brain tissue.Download high-res image (127KB)Download full-size image