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The present study systematically compares the sensitivity and selectivity of the analysis of multiple vitamin D metabolites after chemical derivatization using different reagents for liquid chromatography-tandem mass spectrometry (LC–MS/MS). Generally, chemical derivatization is applied to vitamin D metabolites to increase the ionization efficiency, which is particularly important for very low abundant metabolites. Derivatization can also improve the selectivity of the LC separation. A wide variety of derivatization reagents has been reported in recent years, but information on their relative performance and applicability to different vitamin D metabolites is, unfortunately, not available in the literature. To fill this gap, we investigated vitamin D3, 3β-25-hydroxyvitamin D3 (3β-25(OH)D3), 3α-25-hydroxyvitamin D3 (3α-25(OH)D3), 1,25-dihydroxyvitamin D3 (1,25(OH)2D3), and 24,25-dihydroxyvitamin D3 (24,25(OH)2D3) and compared response factors and selectivity after derivatizing with several important reagents, including four dienophile reagents (4-phenyl-1,2,4-triazoline-3,5-dione (PTAD), 4-[2-(6,7-dimethoxy-4-methyl-3-oxo-3,4-dihydroquinoxalinyl)ethyl]-1,2,4-triazoline-3,5-dione (DMEQ-TAD), Amplifex, 2-nitrosopyridine (PyrNO)) as well as two reagents targeting hydroxyl groups: isonicotinoyl chloride (INC) and 2-fluoro-1-methylpyridinium-p-toluenesulfonate (FMP-TS). In addition, a combination of dienophiles and hydroxyl group reagents was examined. For LC separations, reversed-phase C-18 and mixed-mode pentafluorophenyl HPLC columns using different compositions of the mobile phase were compared. With respect to detection sensitivity, the optimum derivatization reagent for the profiling of multiple metabolites was Amplifex. Nevertheless, FMP-TS, INC, PTAD, or PTAD combined with an acetylation reaction showed very good performance for selected metabolites. These reagent combinations provided signal enhancements on the order of 3- to 295-fold depending on the compound. Chromatographic separation of the dihydroxylated vitamin D3 species was readily achieved using any of the derivatization reactions, while for 25(OH)D3 epimers, only PyrNO, FMP, INC, and PTAD combined with acetylation enabled complete separation. In conclusion, we believe this study can serve as a useful reference for vitamin D laboratories, to help analytical and clinical scientists decide which derivatization reagent to choose for their application.

The determination of vitamin D metabolites as status marker or for diagnostic purposes is almost entirely conducted from blood serum or plasma. Other biological matrices, however, have also interested researchers, for two main reasons: (1) alternative matrices may allow non-invasive sampling, permit easier sample transfer and require less demanding storage conditions; and (2) the levels of vitamin D metabolites in other body compartments may further aid the understanding of vitamin D metabolism and function. Thus, the development of reliable and efficient sample preparation protocols for sample matrices other than serum/plasma, which will remove potential interferences and selectively extract the targeted metabolites, is of great importance. This review summarizes sample preparation methods for measurement of vitamin D metabolites using liquid chromatography-(tandem)mass spectrometry in more than ten different human tissues, including hair, saliva, adipose tissue, brain and others. Graphical abstract

The increasing popularity of high-resolution mass spectrometry has led to many custom software solutions to process, interpret, and reveal information from high-resolution mass spectra. Although there are numerous software packages for peak-picking, calibration, and formula-finding, there are additional layers of information available when it comes to detecting repeated motifs from polymers or molecules with repeating structures or products of chemical or biochemical transformations that exhibit systematic, serial chemical changes of mass. Constellation is an open-source, Python-based web application that allows the user first to expand their high-resolution mass data into the mass defect space, after which a trend finding algorithm is used for supervised or unsupervised detection of repeating motifs. Many adjustable parameters allow the user to tailor their trend-search to target particular chemical moieties or repeating units, or search for all potential motifs within certain limits. The algorithm has a built-in optimization routine to provide a good starting point for the main trend finding parameters before user customization. Visualization tools allow interrogation of the data and any trends/patterns to a highly specific degree and save publication-quality images directly from the interface. As Constellation is deployed as a web application, it is easily used by anyone with a web browser; no software download or high-powered computer is required, as computations are performed on a remote high-powered data server run by our group.

This tutorial review focuses on analytical challenges encountered with the liquid chromatography-tandem mass spectrometry determination of 25-hydroxyvitamin D, which is currently still considered the metabolite that is most representative of vitamin D status. It describes how multiple binding states of circulating 25-hydroxyvitamin D (phase II metabolites, epimers, free/bioavailable/protein-bound species) can influence the accuracy of the analytical determination. It also summarizes important chemical species that can inadvertently contribute to vitamin D status and thus cause systematic errors. These interfering endogenous and exogenous compounds might be isomers of vitamin D, constitutional isomers or isobars and the article outlines techniques to eliminate or minimize these interferences, including chromatographic separations, ion mobility spectrometry, and high-resolution mass spectrometry.

The field of high-resolution mass spectrometry (HRMS) and ancillary hyphenated techniques comprise a rapidly expanding and evolving area. As popularity of HRMS instruments grows, there is a concurrent need for tools and solutions to simplify and automate the processing of the large and complex datasets that result from these analyses. Constellation is one such of these tools, developed by our group over the last two years to perform unsupervised trend detection for repeating, polymeric units in HRMS data of complex mixtures such as natural organic matter, oil, or lignin. In this work, we develop two new unsupervised algorithms for finding chemically-meaningful changing units in HRMS data, and incorporate a molecular-formula-finding algorithm from the open-source CoreMS software package, both demonstrated here in the Constellation software environment. These algorithms are evaluated on a collection of open-source HRMS datasets containing polymeric analytes (PEG 400 and NIST standard reference material 1950, both metabolites in human plasma, as well as a swab extract containing polymers), and are able to successfully identify all known changing units in the data, including assigning the correct formulas. Through these new developments, we are excited to add to a growing body of open-source software specialized in extracting useful information from complex datasets without the high costs, technical knowledge, and processor-demand typically associated with such tools.

The composition of acoustically levitated droplets was probed by a novel combination of mid-IR laser evaporation and subsequent postionization via secondary electrospray ionization. The combination of microliter samples and subnanoliter sampling provided time-resolved interrogation of droplets and allowed for a kinetic investigation of the laser-induced release of the analyte, which was found to strongly depend on the analytes. The observed substance-specific delayed release of the analytes permitted baseline-separated discrimination of the analytes, ideal for the study of complex samples. The additionally applied postionization scheme was found to enable efficient detection of small volatile compounds as well as peptides. The detection of small molecules and peptides occurred under very different sampling geometries, pointing to two distinct underlying ionization mechanisms. Overall, our results suggest that the experimental setup presented in this study can serve as a widely applicable platform to study chemical reactions in acoustically levitated droplets as model reactors.

Liquid chromatography/tandem mass spectrometry (LC-MS/MS) is widely used to determine vitamin D

The development of appropriate analytical screening techniques for pharmaceuticals and personal care products (PPCPs) is the basis for studying the distribution and environmental impact of emerging contaminants (ECs). Mass spectrometry-based screening methods vary with the complexity of the target compounds. It is challenging to balance both positive and negative ion quantification with a low detection limit. To establish a set of experimental methods including extraction, chromatography-separation and mass spectrometry screening is one of the most important topics in PPCP research. This paper describes a universal and efficient qualification and quantification protocol for the simultaneous detection of 34 PPCPs in different environmental samples in a single analytical data acquisition run.Thirty-four representative PPCPs, which are widely distributed in the environment with high ecological toxicity and complex chemical structures, were selected as representative target ECs. The extraction of the target PPCPs was achieved using only one solid-phase extraction cartridge without the need to adjust the pH of samples. The enriched samples were detected by LC-MS/MS in both positive and negative ion modes simultaneously. The protocol was evaluated based on the accuracy, precision, detection limits and matrix effects.This method achieved simultaneous detection of PPCPs in both positive and negative ion modes, with a single analytical cycle of 12 min. The observed SPE recoveries were between 40% and 115%. The instrumental detection limits (IDL) varied from 0.01 to 1 pg, and the method detection limits (MDL) were between 0.002 and 3.323 ng/l in different matrices. Most of the PPCPs were subjected to matrix suppression below 30%. The method was successfully applied for quantitative analysis of the PPCPs in different environmental samples, including river samples, wastewater treatment plant (WWTP) samples and soil samples.This protocol developed a rapid and efficient detection method to simultaneous qualitative and quantitative 34 representative PPCPs in the environment. The IDL ranged from 0.01 to 1 pg and the MDL ranged from 0.002 to 3.323 ng/l in different matrices. The detection limit was one order of magnitude lower compared to previous studies. The protocol also provided a wide application range for different environmental matrices, which permitted the migration and transformation of PPCPs to be explored.

Lignin is currently one of the most promising biologically derived resources, due to its abundance and application in biofuels, materials and conversion to value aromatic chemicals. The need to better characterize and understand this complex biopolymer has led to the development of many different analytical approaches, several of which involve mass spectrometry and subsequent data analysis. This review surveys the most important analytical methods for lignin involving mass spectrometry, first looking at methods involving gas chromatography, liquid chromatography and then continuing with more contemporary methods such as matrix assisted laser desorption ionization and time-of-flight-secondary ion mass spectrometry. Following that will be techniques that directly ionize lignin mixtures—without chromatographic separation—using softer atmospheric ionization techniques that leave the lignin oligomers intact. Finally, ultra-high resolution mass analyzers such as FT-ICR have enabled lignin analysis without major sample preparation and chromatography steps. Concurrent with an increase in the resolution of mass spectrometers, there have been a wealth of complementary data analyses and visualization methods that have allowed researchers to probe deeper into the “lignome” than ever before. These approaches extract trends such as compound series and even important analytical information about lignin substructures without performing lignin degradation either chemically or during MS analysis. These innovative methods are paving the way for a more comprehensive understanding of this important biopolymer, as we seek more sustainable solutions for our human species’ energy and materials needs.

A combination of acoustic levitation, laser vaporization, and atmospheric pressure chemical ionization mass spectrometry (APCI-MS) is presented in this study that enabled sensitive analysis of pharmaceutical drugs from an aqueous sample matrix. An unfocused pulsed infrared laser provided contactless sample desorption from the droplets trapped inside an acoustic levitator by activation of the OH stretching band of aqueous and alcoholic solvents. Subsequent atmospheric pressure chemical ionization was used between the levitated droplet and the mass spectrometer for postionization. In this setup, the unfocused laser gently desorbed the analytes by applying very mild repulsive forces. Detailed plume formation studies by temporally resolved schlieren experiments were used to characterize the liquid gas transition in this process. In addition, the role of different additives and solvent composition was examined during the ionization process. The analytical application of the technique and the proof-of-concept for quantitative analysis were demonstrated by the determination of selected pharmaceutical drugs in aqueous matrix with limits of quantification at the lower nanomolar level and a linear dynamic range of 3-4 orders of magnitude.

Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR MS) combined with different ionization techniques provides a powerful means to characterize dissolved organic matter (DOM) at the molecular level. Solid-phase extraction (SPE) is currently the most widely utilized method for extracting the DOM, but one-step elution using methanol does not provide a comprehensive picture of DOM. The development of efficient extraction and enrichment methods as well as characterization techniques from water samples remains a priority for DOM research, which is investigated in this study.The DOM was extracted from lake water by SPE using one-step elution (methanol or dichloromethane) and multistep elution (water, methanol, acetone, and dichloromethane). A combination of electrospray ionization (ESI), atmospheric pressure photoionization (APPI), and matrix-free laser desorption ionization (LDI) was utilized for FT-ICR MS analysis in both positive (+) and negative (-) ion modes.The total recovery of the multistep elution was 23.5% higher as compared to the investigated one-step elution procedure (85% vs. 61.6%); however, a comparison of the observed molecular species and the range of diversity under different ionization techniques along with the statistical analyses showed that proper selection of solvent and ionization method was required to explore specific compounds from the sample.For DOM species containing different heteroatoms, a combination of ESI, APPI, and LDI can offer a comprehensive profile of DOM in aquatic ecosystems. The specific molecular formulae of each ionization technique are characterized as follows: ESI- mode exhibited strong selectivity for lignin-like and tannins-like species with high oxygen content, as well as organosulfates. ESI+ favored lipid species and peptide/protein compounds. Unsaturated and condensed aromatic hydrocarbons with low oxygen were preferably ionized by both APPI and LDI.

Exploring the source, transformation pathways, and the fate of natural organic matter (NOM) is critical to understanding the regional/global carbon cycle and carbon budget. The dissolved fraction of NOM, i.e., dissolved organic matter (DOM), is a complex mixture resulting from the transformation of plant, animal and microbial matter and plays a crucial role in many biogeochemical processes at the land-ocean-atmosphere interfaces. The advance of Fourier transform ion cyclotron resonance mass spectrometer (FT-ICR MS) makes the detailed characterization of DOM at the molecular level possible. On the other hand, elucidation of complex DOM sample also presents significant analytical challenges, and these challenges also act as a driving force for the instrumentation and methodology development on FT-ICR MS. This review article has been written to aid those working in biogeochemistry, environmental and atmospheric chemistry, and related areas which investigate elemental cycles and DOM transformations. First, the fundamental theory, historical perspective, and recent advances in the field have been introduced. The detailed molecular characterization of environmental and geological samples continues to present significant analytical challenges, and it also has become a driving force for the development of the instrumentation and experimental methods. These achievements in DOM analysis have had an impact upon the fields of environmental science, geochemistry, and analytical chemistry. Next, varieties of applications of FT-ICR MS have also been described, followed by our view of the future of this technique in earth science research. We believe that this review covers the essential pairing of FT-ICR MS and collectively offers environmental and geochemical scientists a substantial resource for their research. Graphical abstract

Vitamin D compounds are a group of secosteroids derived from cholesterol that are vital for maintaining bone health in humans. Recent studies have shown extraskeletal effects of vitamin D, involving vitamin D metabolites such as the dihydroxylated vitamin D

The determination of vitamin D metabolites as status marker or for diagnostic purposes is almost entirely conducted from blood serum or plasma. Other biological matrices, however, have also interested researchers, for two main reasons: (1) alternative matrices may allow non-invasive sampling, permit easier sample transfer and require less demanding storage conditions; and (2) the levels of vitamin D metabolites in other body compartments may further aid the understanding of vitamin D metabolism and function. Thus, the development of reliable and efficient sample preparation protocols for sample matrices other than serum/plasma, which will remove potential interferences and selectively extract the targeted metabolites, is of great importance. This review summarizes sample preparation methods for measurement of vitamin D metabolites using liquid chromatography-(tandem)mass spectrometry in more than ten different human tissues, including hair, saliva, adipose tissue, brain and others.

Liquid chromatography/tandem mass spectrometry is firmly established today as the gold standard technique for analysis of vitamin D, both for vitamin D status assessments as well as for measuring complex and intricate vitamin D metabolic fingerprints. While the actual mass spectrometry technology has seen only incremental performance increases in recent years, there have been major, very impactful changes in the front- and back-end of MS-based vitamin D assays; for example, the extension to new types of biological sample matrices analyzed for an increasing number of different vitamin D metabolites, novel sample preparation techniques, new powerful chemical derivatization reagents, as well the continued integration of high resolution mass spectrometers into clinical laboratories, replacing established triple-quadrupole instruments. At the same time, the sustainability of mass spectrometry operation in the vitamin D field is now firmly established through proven analytical harmonization and standardization programs. The present review summarizes the most important of these recent developments.

Overcoming the detrimental effects of sweet spots during crystallization is an important step to improve the quantitative abilities of matrix‐assisted laser desorption/ionization (MALDI) mass spectrometry. In this study, we introduce MALDI targets, which exhibit a channel design to reduce sweet spot phenomena and improve reproducibility. The size of the channels was 3.0 mm in length, 0.35 mm in depth, and 0.40 mm in width, adjusted to the width of the implemented laser beam. For sample deposition, the matrix/sample mixture was homogenously deposited into the channels using capillary action. To demonstrate the proof‐of‐principle, the novel plates were used for the quantification of acetyl‐L‐carnitine in human blood plasma using a combined standard addition and isotope dilution method. The results showed that the reproducibility of acetyl‐L‐carnitine detection was highly improved over a conventional MALDI‐MS assay, with RSD values of less than 5.9% in comparison with 15.6% using the regular MALDI method. The limits of quantification using the new plates were lowered approximately two‐fold in comparison with a standard rastering approach on a smooth stainless‐steel plate. Matrix effects were also assessed and shown to be negligible. The new assay was subsequently applied to the quantification of acetyl‐L‐carnitine in human plasma samples.

The sources and composition of dissolved organic matter (DOM) in rivers are critical to water quality and aquatic ecosystems. Studies on detailed composition of organic matter in rivers in the farming-pastoral ecotone are relatively limited in the research community. To better understand the characteristics and dynamics of DOM, Yang River in North China was selected as the study area because of its profound influences on the farming-pastoral ecotone nearby. A combination of fluorescence spectroscopy and Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR MS) techniques revealed that the DOM composition of Yang River is driven by land use. DOM in Yang River is predominantly imported from allochthonous inputs, together with agricultural runoff, pastureland, and urban sewage, causing a comprehensive impact on DOM. In detail, DOM associated with cropland inputs was dominated by lignin-like species, with higher nitrogen content. In comparison, DOM related to grassland is more diverse and susceptible to degradation. An increase in urban areas led to an increase in sulfur-containing compounds, while their oxygen, nitrogen, and aromaticity contents were significantly lower than those in cropland. Interestingly, urban-influenced lignin-like compounds may be associated with the effluents from the pulp and paper mill. Additionally, synthetic surfactants from the lower section of the river were also structurally identified by tandem mass spectrometry. Overall, this study could provide valuable insights into the DOM sources and their transformation dynamics at a molecular level, which could be an indicator for riverine water quality management and be applied to other farming-pastoral ecotones straightforward.

The metabolism of vitamin D

A series of ultrathin, homogenous gold nanoparticle (AuNP) substrates for surface-assisted laser desorption/ionization mass spectrometry (SALDI-MS) were prepared using a simple air/water interface approach. These SALDI substrates enabled soft ionization and provided significant improvements in terms of signal intensities and reduced background levels in comparison to other AuNP morphologies for different analytes such as fatty acids, peptides, amino acids, saccharides, and drugs. Through different microscopic and spectroscopic methods, we determined that the packing homogeneity of the [AuNP]

LC-MS/MS is widely utilized today for quantification of vitamin D in biological fluids. Mass spectrometric assays for vitamin D require very careful method optimization for precise and interference-free, accurate analyses however. Here, we explore chemical derivatization and matrix-assisted laser desorption/ionization (MALDI) as a rapid alternative for quantitative measurement of 25-hydroxyvitamin D3 in human serum, and compare it to results from LC-MS/MS. The method implemented an automated imaging step of each MALDI spot, to locate areas of high intensity, avoid sweet spot phenomena, and thus improve precision. There was no statistically significant difference in vitamin D quantification between the MALDI-MS/MS and LC-MS/MS: mean ± standard deviation for MALDI-MS—29.4 ± 10.3 ng/mL—versus LC-MS/MS—30.3 ± 11.2 ng/mL (P = 0.128)—for the sum of the 25-hydroxyvitamin D epimers. The MALDI-based assay avoided time-consuming chromatographic separation steps and was thus much faster than the LC-MS/MS assay. It also consumed less sample, required no organic solvents, and was readily automated. In this proof-of-concept study, MALDI-MS readily demonstrated its potential for mass spectrometric quantification of vitamin D compounds in biological fluids.

Octocrylene (OC) is an emerging UV filter, which is used in the majority of sunscreens as well as other personal care products (PCP) and consumer products. Its presence in various environmental matrices has been reported. However, information on the internal OC exposure in humans is not available, due to the lack of appropriate biomarkers of exposure and analytical methods. Here, we describe a rugged, precise, and accurate analytical method for the determination of three OC metabolites (ester hydrolysis and alkyl chain oxidation products) in human urine by stable isotope dilution analysis. Urine samples are incubated with β-glucuronidase (E. coli K12) and then analyzed by liquid chromatography-electrospray ionization-triple quadrupole-tandem mass spectrometry with online turbulent flow chromatography for sample cleanup and analyte enrichment (online-SPE-LC-MS/MS). Syntheses of analytical standards, including deuterium-labeled internal standards, are also described. In a pilot study, we investigated the applicability of the metabolites as biomarkers of exposure in urine samples from the general population (n = 35). OC metabolites were detected in 91% of the samples, with the highest concentrations for three individuals having used sunscreen within 5 days prior to sample collection. We will apply the method in future human biomonitoring studies for OC exposure and risk assessment.

Ambient ionization techniques are typically used to analyze samples in their native states with minimal or no sample pretreatment prior to mass spectrometric (MS) analysis. Desorption electrospray ionization (DESI) and direct analysis in real time (DART) were systematically investigated in this work for direct solid analysis of depolymerized lignin samples, with the goal of rapidly fingerprinting these samples, to efficiently characterize the subunits of this renewable energy source. High resolution MS was required for enhanced selectivity in this study due to the inherent structural complexity of lignin. DESI provided results across a broader mass range (up to m/z 700) than DART and also ionized saturated compounds of low oxygen-to-carbon (O/C) ratios and low double bond equivalents (DBE). While DART detected the same core lignin monomeric and dimeric compounds as seen with DESI and electrospray ionization (ESI), results were restricted to a narrower mass range to m/z 500, due to thermal degradation and losses of methoxy groups. In contrast to DESI and ESI, the DART spectra were nearly void of saturated components. On a structural level, the core lignin compounds were visually fingerprinted and ionization method performances critically assessed by employing simplified Kendrick-based data mining approaches. A novel simplified data visualization approach was developed in this work based on modified Kendrick mass defect (KMD) filtering for lignin subunits and plotting the mass defect values against the degree of unsaturation. Direct visualization of monomeric, dimeric and trimeric lignin species was simplified by the KMD separation plots, easily allowing the core lignin compounds to be visually identified and compared. Modified KMD bases, namely methoxy and phenol bases, which represent monomer-specific lignin constituents, were successfully used to classify and group the complex mixture of lignin species. Further separation of methoxy-related lignin species was successfully achieved by employing the more specific phenolic KMD base.

Lignin is the second most abundant natural polymer and a promissing feedstock for the generation of renewable aromatic chemicals. We present an fundamental approach for the electrocatalytic cleavage of lignin dissolved in a recoverable, inexpensive ionic liquid using mixed metal oxide electrodes of different compositions. The distribution of depolymerization products generated by electrochemical oxidation were analyzed by means of mass spectrometry. The distribution and yield of the cracked species was found to depended strongly on the implemented metal catalyst and therefore offers the potential to tailor the amount and composition of the low molecular weight cleavage products. This approach could help to provide a more sustainable valorization of lignin for the potential production of high value aromatic compounds due to synergistic effects.

Vitamin D compounds are secosteroids, which are best known for their role in bone health. More recent studies have shown that vitamin D metabolites and catabolites such as dihydroxylated species (e.g., 1,25- and 24,25-dihydroxyvitamin D3) play key roles in the pathologies of various diseases. Identification of these isomers by mass spectrometry is challenging and currently relies on liquid chromatography, as the isomers exhibit virtually identical product ion spectra under collision induced dissociation conditions. Here, we developed a simple MALDI-CID method that utilizes ion activation of reactive analyte/matrix adducts to distinguish isomeric dihydroxyvitamin D3 species, without the need for chromatography separation or chemical derivatization techniques. Specifically, reactive 1,5-diaminonaphthalene adducts of dihydroxyvitamin D3 compounds formed during MADI were activated and specific cleavages in the secosteroid’s backbone structure were achieved that produced isomer-diagnostic fragment ions.

This experiment describes a simple protocol for teaching acid–base titrations using potentiometry, conductivity, and/or photometry to determine end points without an added indicator. The chosen example examines the titratable acidity of a red wine with NaOH. Wines contain anthocyanins, the colors of which change with pH. Importantly, at the equivalence point, anthocyanins maintain their color, and this effect can be captured optically using a desktop scanner. RGB-based color values are obtained from the digitized images using a dedicated application and employed to generate plots of |v| versus titrant volume. The end point is the point at which the slope (d|v|/Δ/dV) of the titration curve is at a maximum; (d|v| is a vector expression of the color change). The photometric titrations were carried out using a plastic 96-well immunology plate and a flat-bed scanner, and RGB values were extracted simultaneously from all 96 wells in less than 5 min from the images using the ImageJ plugin “ReadPlate”. The wine w...

Matrix-assisted laser desorption/ionization (MALDI)-mass spectrometry imaging (MSI) permits label-free in situ analysis of chemical compounds directly from the surface of two-dimensional biological tissue slices. It links qualitative molecular information of compounds to their spatial coordinates and distribution within the investigated tissue. MALDI-MSI can also provide the quantitative amounts of target compounds in the tissue, if proper calibration techniques are performed. Obviously, as the target molecules are embedded within the biological tissue environment and analysis must be performed at their precise locations, there is no possibility for extensive sample clean-up routines or chromatographic separations as usually performed with homogenized biological materials; ion suppression phenomena therefore become a critical side effect of MALDI-MSI. Absolute quantification by MALDI-MSI should provide an accurate value of the concentration/amount of the compound of interest in relatively small, well-defined region of interest of the examined tissue, ideally in a single pixel. This goal is extremely challenging and will not only depend on the technical possibilities and limitations of the MSI instrument hardware, but equally on the chosen calibration/standardization strategy. These strategies are the main focus of this article and are discussed and contrasted in detail in this tutorial review. This article is part of a Special Issue entitled: MALDI Imaging, edited by Dr. Corinna Henkel and Prof. Peter Hoffmann.

The previously unknown sequences of several pyoverdines (PVD) produced by a biotechnologically-relevant bacterium, namely, Pseudomonas taiwanensis VLB120, were characterized by high performance liquid chromatography (HPLC)-high resolution mass spectrometry (HRMS). The same structural characterization scheme was checked before by analysis of Pseudomonas sp. putida KT2440 samples with known PVDs. A new sample preparation strategy based on solid-phase extraction was developed, requiring significantly reduced sample material as compared to existing methods. Chromatographic separation was performed using hydrophilic interaction liquid chromatography with gradient elution. Interestingly, no signals for apoPVDs were detected in these analyses, only the corresponding aluminum(III) and iron(III) complexes were seen. The chromatographic separation readily enabled separation of PVD complexes according to their individual structures. HPLC-HRMS and complementary fragmentation data from collision-induced dissociation and electron capture dissociation enabled the structural characterization of the investigated pyoverdines. In Pseudomonas sp. putida KT2240 samples, the known pyoverdines G4R and G4R A were readily confirmed. No PVDs have been previously described for Pseudomonas sp. taiwanensis VLB120. In our study, we identified three new PVDs, which only differed in their acyl side chains (succinic acid, succinic amide and malic acid). Peptide sequencing by MS/MS provided the sequence Orn-Asp-OHAsn-Thr-AcOHOrn-Ser-cOHOrn. Of particular interest is the presence of OHAsn, which has not been reported as PVD constituent before.

While the biological function of the 3α epimer of 25-hydroxyvitamin D 3 (25(OH)D 3 ) remains unknown, its presence needs to be accurately captured and separated from the main 3β epimer, to avoid positive bias in vitamin D status analyses. Several recent LC-MS/MS assays for 25(OH)D 3 successfully separate the 3α and 3β epimers by chromatography. Unfortunately, none of the existing LC-MS/MS assays, which utilize dried blood spots (DBS) as sampling/storage vessels, is able to quantify the individual epimers. DBS are often used for analysis of infant blood, however, and these samples are particularly likely to contain significant levels of interfering 3α epimer. Furthermore, proper calibration of DBS samples is much more difficult to achieve than for liquid serum or plasma samples. We addressed this important issue by creating an artificial vitamin D -free whole blood for calibration and then quantified 3α- and 3β-25(OH)D 3 levels from DBS. After chemical derivatization, the vitamin D epimers were separated on a PFP column and concentrations determined by electrospray ionization LC-MS/MS on a triple quadrupole mass spectrometer. Calibration with artificial whole blood showed improved precision over standard addition (7.6 versus 31.5% RSD for 3β-25(OH)D 3 ). The limits of quantification for 3β-25(OH)D 3 and for 3α-25(OH)D 3 were 1.0 and 0.1 ng/mL, respectively. Excellent intra/interday precisions between 2.1 and 2.2% CV (intra) and 4.4–5.3% CV (inter) were established for 3β-25(OH)D 3 and 3α-25(OH)D 3 . For 3β-25(OH)D 3 , only small concentration-independent bias and deviation of 3 showed deviations of

Lignin is a highly complex, plant-derived natural biomass component, the analysis of which requires significant demands on the analytical platform. Fourier transform ion cyclotron mass spectrometry (FT-ICR MS) has been shown to be able to readily assess the complexity of lignin and lignin degradation products by assigning tens of thousands of compounds with elemental formulae. Nevertheless, many experimental and instrumental parameters introduce discrimination towards certain components, which limits the comprehensive MS analysis. As a result, a complete characterization of the lignome remains a challenge. The present study investigated a degraded lignin sample using FT-ICR MS and compared several atmospheric pressure ionization methods, e.g., electrospray ionization, atmospheric-pressure chemical ionization, and atmospheric-pressure photoionization. The results clearly show that the number of heteroatoms (e.g., N, S, P) in the sample greatly increases the chemical diversity of lignin, while at the same time also providing potentially useful biomarkers. We demonstrate here that FT-ICR MS was able to directly isolate isotopically pure single components from the ultra-complex mixture for subsequent structural analysis, without the time-consuming chromatographic separation. CAPSULE: Various ionization techniques coupled to FT-ICR MS provide a powerful tool to assess the lignome coverage.

Among the different techniques for mass analysis, ultra‐high‐resolution Fourier transform ion cyclotron resonance (FTICR) is the method of choice for highly complex samples, as it offers unrivaled mass accuracy and resolving power, combined with a high degree of flexibility in hybrid instruments as well as for ion activation techniques. FTICR instruments are readily embraced by the biological and biomedical research communities and applied over a wide range of applications for the analysis of biomolecules such as carbohydrates, lipids, nucleic acids, and proteins. In the field of natural organic matter (NOM) analysis, petroleum‐related studies currently dominate FTICR‐MS applications. Recently, however, there is a growing interest in developing high‐performance MS methods for the characterization of NOM samples from natural aquatic and terrestrial environments. Here, we present an overview of FTICR‐MS techniques for complex, non‐petroleum NOM samples, including data analysis and novel tandem mass spectrometry (MS/MS) methods for structural classifications. © 2020 The Authors. Mass Spectrometry Reviews published by John Wiley & Sons Ltd.

Objective The aim of this study was to determine chlorhexidine retention in different oral sites after a one-time 30 s mouth rinsing. Design Five volunteers were asked to rinse their mouth with 10 ml of 0.2 % chlorhexidine digluconate for 30 s. After rinsing, samples were collected from the interdental area, buccal dental pellicle, anterior labial and posterior buccal mucosa, and saliva with a microbrush at five-time points within 24 h. Retention of chlorhexidine was measured using matrix-assisted laser desorption/ionization-time of flight mass spectrometry with a quantification limit of 15 ng/ml. Results Chlorhexidine remained in the oral cavity at micrograms per milliliter levels for 11 h after mouth rinsing and was even detected 24 h after application. The results showed a distinct decline of intraoral chlorhexidine levels during the first 6 h after rinsing and it was then retained at low concentrations for at least 24 h. Conclusions The dental pellicle and oral mucosa were favorable sites for chlorhexidine retention. The novel method used for chlorhexidine determination offered excellent quantification limits and readily permitted quantification of chlorhexidine.

Lignin is the most abundant natural resource of aromatic moieties and the second most abundant natural biopolymer. Analytical techniques that obtain as much information as possible on the exact structural content of lignin species are essential for developing efficient processes that transform highly complex lignin wastes into value chemicals and biofuels. For mass spectrometric analysis of lignin samples, usually electrospray ionization, atmospheric pressure chemical ionization, or atmospheric pressure photoionization are used as ionization techniques. Matrix-assisted laser desorption/ionization (MALDI) is less frequently applied but offers a much more rapid screening option for lignin mixtures. In this study, we compared several common MALDI matrices for analysis of alkali lignin and discovered that different chemical matrices exhibited very different ionization efficiencies and selectivity with respect to the structures of the lignin-related compounds as well as the presence of heteroatoms. Importantly, the results highlight that the choice of matrix strongly determines the analytical coverage of molecular species in the complex lignin degradation mixtures. Graphical abstract.

Vitamin D measurements in biological fluids by mass spectrometry are challenging at very low concentration levels. As a result, chemical derivatization is often employed to enhance the ionization properties of low abundant vitamin D compounds. Cookson-type reagents such as 4-phenyl-1,2,4-triazoline-3,5-dione (PTAD) or similar derivatives work well but require careful, water-free experimental conditions, as traces of water inactivate the reagent and inhibit or stop the derivatization reactions, thus making quantitative measurements in aqueous samples impossible. We describe a novel electrospray liquid chromatography-tandem mass spectrometry (LC-MS/MS) assay for determining 25-hydroxyvitamin D3 (25(OH)D3) directly in aqueous cellular systems using a new derivatization reagent, the ionic liquid 12-(maleimidyl)dodecyl-tri-n-butylphosphonium bromide (MDBP). The proof-of-concept for the MDBP assay was demonstrated by measuring the levels of 25(OH)D3 in four different human cell types, namely T cells, helper T cells, B cells, and macrophages. In addition to the ability to determine the levels of 25(OH)D3 directly in aqueous samples, the cellular integrity was maintained in our application. We show the time-dependent uptake of 25(OH)D3 into the investigated cells to demonstrate the applicability of the new label. Furthermore, the MDBP derivatization technique may be equally useful in imaging mass spectrometry, where it could be used for response enhancements of spatially localized vitamin D metabolites on wet tissue surfaces, without destroying the integrity of the tissue surface.

Recently, hepatic immaturity was cited as a possible reason for high levels of the C-3 epimer of 25-hydroxyvitamin (25(OH)D) in premature infants: however what role, if any, the liver plays in controlling epimer concentrations is unknown. This study assesses 3-epi-25-hydroxyvitamin D (3-epi-25(OH)D) levels during the course of cholecalciferol supplementation in adults with chronic liver diseases (CLD). Vitamin D metabolites were analyzed in 65 CLD patients with 25(OH)D

First impressions are very important and, when these are negative, they can adversely affect a manuscript's journey through the scientific publication system. This short guide highlights some crucial factors to take into consideration before submitting a manuscript for review in a scientific journal. The aim is to advise authors on the best way to present their research, to comply with formal requirements of a journal and to optimize the first impression made. Copyright © 2016 John Wiley & Sons, Ltd.

In this study, we report p-coumaric acid as novel and effective response marker for indirectly measuring the levels of hypoxia in normal primary bronchial epithelial cells. We developed a simple and rapid technique based on hydrophilic interaction chromatography-electrospray ionization-mass spectrometry (HILIC-ESI-MS). During 168h of hypoxia without induction of reactive oxygen species (ROS), an almost linear increase of p-coumaric acid levels was observed. We interpret the increasing p-coumaric acid concentrations during hypoxia as a result of cell damage, triggered by reduced co-enzyme Q10 levels, because the oxidative cascade was not able to supply sufficient energy. The HILIC-ESI-MS assay within p-coumaric acid exhibited a linear dynamic range from 60 to 610 ng/μL with correlation coefficient of 0.9998. The precision of the assay was ≤15% RSD and method accuracies between 97 and 108%.

Gas phase ion/molecule reactions are often used in analytical applications to support the analysis of isomers or to identify specific functional groups of organic molecules. Until now, deliberate chemical reactions have not been performed in differential ion mobility spectrometry (DMS) devices except for hydrogen exchange and cluster formation. The present work extends that of Colorado and Brodbelt (Anal Chem 66:2330-5, 1994) on ion/molecule reactions in an ion trap mass spectrometer. In this study, class-specific chemical reactions of 4-quinolone antibiotics with various chemical reagents were used to demonstrate the analytical utility of ion/molecule reactions in a DMS drift cell. For these reactions, dehydrated reactive precursor ions were initially formed and made to undergo annulation reactions with selected reagents within the timescale of the DMS separation. Careful study of the energies required for dissociation of the adducts confirmed the covalent nature of the newly formed bond; thus demonstrating the analytical utility of this approach. Graphical abstract.

Multiple sclerosis (MS) is an immune-mediated neurodegenerative disease of the central nervous system (CNS). One of the most promising recent medications for MS is teriflunomide. Its primary mechanism of action is linked to effects on the peripheral immune system by inhibiting dihydroorotate dehydrogenase (DHODH)-catalyzed de novo pyrimidine synthesis and reducing the expansion of lymphocytes in the peripheral immune system. Some in vitro studies suggested, however, that it can also have a direct effect on the CNS compartment. This potential alternative mode of action depends on the drug's capacity to traverse the blood-brain barrier (BBB) and to exert an effect on the complex network of brain biochemical pathways. In this paper, we demonstrate the application of high-resolution/high-accuracy matrix-assisted laser desorption/ionization Fourier-transform ion cyclotron resonance mass spectrometry for molecular imaging of the mouse brain coronal sections from animals treated with teriflunomide. Specifically, in order to assess the effect of teriflunomide on the mouse CNS compartment, we investigated the feasibility of teriflunomide to traverse the BBB. Secondly, we systematically evaluated the spatial and semi-quantitative brain metabolic profiles of 24 different endogenous compounds after 4-day teriflunomide administration. Even though the drug was not detected in the examined cerebral sections (despite the high detection sensitivity of the developed method), in-depth study of the endogenous metabolic compartment revealed noticeable alterations as a result of teriflunomide administration compared to the control animals. The observed differences, particularly for purine and pyrimidine nucleotides as well as for glutathione and carbohydrate metabolism intermediates, shed some light on the potential impact of teriflunomide on the mouse brain metabolic networks. Graphical Abstract.

Lignin is the second most abundant natural biopolymer and its wastes are significant sources for renewable chemicals as an alternative to conventional fossil fuels. Consequently, chemical characterization methods are required to assess the content of valuable chemicals contained in these complex lignin wastes. This short overview summarizes rapid data-processing methods developed in our laboratory for application to full-scan raw data from high-resolution mass spectrometry experiments of decomposed lignin samples. The discussed graphical and statistical methods support the initial classification and elucidation of the main structural features of the lignin components without the need for time-consuming tandem mass spectrometry analyses.