Surface-Assisted Laser Desorption/Ionization Imaging Mass Spectrometry (SALDI-IMS)-Based Detection of Vinca Alkaloids Distribution in the Petal of Madagascar Periwinkle.

The surface-assisted laser desorption/ionization (SALDI) technique uses inorganic materials to aid desorption and ionization of molecules. SALDI is suitable for analyzing small molecules due to the absence of interfering signals in the low m/z range originating from the organic matrix. Imaging mass spectrometry (IMS) is a versatile imaging approach with high spatial resolution for analyzing various molecular species, but its application depends heavily on the ionization method. We have developed a functionalized titanium dioxide (TiO2) nanowire as a solid substrate for SALDI-MS detection of low-molecular-weight molecules. We apply this novel substrate for imprinting fragile specimens such as petals and further SALDI-IMS analysis. The TiO2 nanowire substrate is prepared from a commercial Ti plate by a hydrothermal process and subsequently chemically modified to improve the quality and selectivity of imprinting as well as the sensitivity of SALDI-IMS analysis. Here, the functionalized TiO2 nanowire substrate is applied to visualize the distribution of vinca alkaloids in the petal of Madagascar periwinkle (Catharanthus roseus).

Visualizing vinca alkaloids in the petal of Catharanthus roseus using functionalized titanium oxide nanowire substrate for surface-assisted laser desorption/ionization imaging mass spectrometry.

Imaging mass spectrometry (IMS) is a powerful technique that enables analysis of various molecular species at a high spatial resolution with low detection limits. In contrast to the matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) approach, surface-assisted laser desorption/ionization (SALDI) can be more effective in the detection of small molecules due to the absence of interfering background signals in low m/z ranges. We developed a functionalized TiO2 nanowire as a solid substrate for IMS of low-molecular-weight species in plant tissues. We prepared TiO2 nanowires using an inexpensive modified hydrothermal process and subsequently functionalized them chemically with various silane analogs to overcome the problem of superhydrophilicity of the substrate. Chemical modification changed the selectivity of imprinting of samples deposited on the substrate surface and thus improved the detection limits. The substrate was applied to image distribution of the metabolites in very fragile specimens such as the petal of Catharanthus roseus. We observed that the metabolites are distributed heterogeneously in the petal, which is consistent with previous results reported for the C. roseus plant leaf and stem. The intermediates corresponding to the biosynthesis pathway of some vinca alkaloids were clearly shown in the petal. We also performed profiling of petals from five different cultivars of C. roseus plant. We verified the semi-quantitative capabilities of the imprinting/imaging approach by comparing results using the LC-MS analysis of the plant extracts. This suggested that the functionalized TiO2 nanowire substrate-based SALDI is a powerful technique complementary to MALDI-MS.

Temporal Correlations of Skin and Blood Metabolites with Clinical Outcomes of Biologic Therapy in Psoriasis.

BACKGROUND: Psoriasis is an inflammatory skin disease causing multisystem effects. Introduction of biologic drugs has led to promising results in treatment of this disease. Here, we carry out time-dependent profiling of psoriasis-related putative metabolic biomarkers. METHODS: Skin excretion specimens were collected from 17 patients with psoriasis treated with biologics for 7 months. Blood specimens were obtained from the same patients at intervals of 1-3 months. A hydrogel micropatch sampling technique was implemented to collect lesional (L) and nonlesional (NL) skin specimens. The collected skin and blood specimens were analyzed by mass spectrometric methods. RESULTS: The metabolites present on L skin-in particular, choline, and citrulline-showed greater dynamics, corresponding to the resolution of psoriasis than the metabolites present in NL skin or blood. Choline levels in L skin and blood correlated positively, while citrulline correlated negatively with the severity of individual psoriasis plaques and general disease severity, respectively. Nevertheless, the correlations between the metabolite levels in blood and general disease severity were weaker than those between the metabolite levels on L skin and severity of individual plaques. The changes of these skin metabolites were more prominent in the responders to the treatment than in the nonresponders. CONCLUSIONS: The results support the feasibility of characterizing dynamic changes in psoriatic skin metabolic profiles with the hydrogel micropatch probes and mass spectrometric tests. The study represents one of few attempts to explore relationships between skin and blood metabolite concentrations. However, practical use of the methodology in close clinical monitoring is yet to be demonstrated.

Predicting Breast Cancer by Paper Spray Ion Mobility Spectrometry Mass Spectrometry and Machine Learning.

Paper spray ionization has been used as a fast sampling/ionization method for the direct mass spectrometric analysis of biological samples at ambient conditions. Here, we demonstrated that by utilizing paper spray ionization-mass spectrometry (PSI-MS) coupled with field asymmetric waveform ion mobility spectrometry (FAIMS), predictive metabolic and lipidomic profiles of routine breast core needle biopsies could be obtained effectively. By the combination of machine learning algorithms and pathological examination reports, we developed a classification model, which has an overall accuracy of 87.5% for an instantaneous differentiation between cancerous and noncancerous breast tissues utilizing metabolic and lipidomic profiles. Our results suggested that paper spray ionization-ion mobility spectrometry-mass spectrometry (PSI-IMS-MS) is a powerful approach for rapid breast cancer diagnosis based on altered metabolic and lipidomic profiles.

Ambient Ionization Mass Spectrometry Today and Tomorrow: Embracing Challenges and Opportunities.

Ambient ionization mass spectrometry (AIMS) has grown into a group of emerging analytical techniques that allow rapid, real-time, high-throughput, in situ, and in vivo analysis in many scientific fields including biomedicine, pharmaceuticals, and forensic sciences. While dozens of AIMS techniques have been introduced over the past two decades, their broad commercial and industrial use is still restricted by multiple challenges. In this Perspective, we discuss the most relevant technical challenges facing AIMS, i.e., reproducibility, quantitative ability, molecular coverage, sensitivity, and data complexity, and scientists' recent attempts to overcome these hurdles. Furthermore, we present future directions of AIMS from our perspective, including the necessity that efforts should be made to unravel blind biomolecules in routine analysis, the construction of a data depository for AIMS users, the full automation of pipelines for prospect integration in a robotic laboratory, the movement toward on-site tests, and the expansion of outreach to motivate government officials in policymaking. We anticipate that, with progress in these critical but immature areas, AIMS technology will keep evolving to become a more robust and user-friendly set of technologies and, consequently, be translated into everyday life practice.

Analytical methods for cholesterol quantification.

Cholesterol is an important lipid molecule in cell membranes and lipoproteins. Cholesterol is also a precursors of steroid hormones, bile acids, and vitamin D. Abnormal levels of cholesterol or its precursors have been observed in various human diseases, such as heart diseases, stroke, type II diabetes, brain diseases and many others. Therefore, accurate quantification of cholesterol is important for individuals who are at increased risk for these diseases. Multiple analytical methods have been developed for analysis of cholesterol, including classical chemical methods, enzymatic assays, gas chromatography (GC), liquid chromatography (LC), and mass spectrometry (MS). Strategy known as ambient ionization mass spectrometry (AIMS), operating at atmospheric pressure, with only minimal sample pretreatments for real time, in situ, and rapid interrogation of the sample has also been employed for quantification of cholesterol. In this review, we summarize the most prevalent methods for cholesterol quantification in biological samples and foods. Nevertheless, we highlight several new technologies, such as AIMS, used as alternative methods to measure cholesterol that are potentially next-generation platforms. Representative examples of molecular imaging of cholesterol in tissue sections are also included in this review article.

Kinetic study of continuous liquid-liquid extraction of wine with real-time detection.

Kinetic optimization of continuous liquid-liquid extraction (CLLE) can shorten sample preparation times and reduce losses of labile or volatile analytes. Here, we coupled a downscaled CLLE apparatus with atmospheric pressure chemical ionization interface of triple quadrupole mass spectrometer. Real-time sampling was guided by an Arduino-based programmable logic controller. The recorded datasets were processed to compute the extraction rate constants for the target analytes. The extraction time in subsequent on-line experiments was set to 180 min as a compromise between the reduction of the analysis time and maximizing its yield. Interestingly, off-line analysis of the extract produced different results than on-line analysis pointing to the immanent degradation of the collected extract aliquots. Next, we implemented this hyphenated system in the analysis of red wine samples, which were stored during different periods of time after opening the bottle. The results reveal differences in the depletion of the volatile wine components during storage.

Cool Mist Scavenging of Gas-Phase Molecules.

The purpose of analytical extractions is to simplify sample matrix without losing analyte molecules. Here we present a technique of extracting volatile compounds by scavenging gas-phase molecules with tiny liquid droplets (<10 µm). A cool mist of the extracting solvent is generated by an ultrasonic transducer, transferred to the headspace of the sample chamber under atmospheric conditions, and pushed by a small pressure difference toward a condenser. By slowly passing over the sample, the microdroplets extract volatile species present in the sample headspace, and they coalesce in a cooled zone. The condensed liquid is collected for analysis by direct infusion mass spectrometry or chromatography coupled with mass spectrometry. Due to the high surface-to-volume ratio of the microdroplets, the mist depletes a great share of the volatile organics present in the headspace. Other advantages of cool mist scavenging include: selective extraction of gas-phase molecules, the extracting solvent can be miscible with the sample solvent, simplicity, high speed, and no requirement for heating that could potentially decompose the sample. In this study, cool mist scavenging was first tested on artificial samples containing esters. The relationship between the sample concentration and the extract concentration was verified theoretically and experimentally. Some of the possible confounding effects were tested and discussed. The technique was subsequently applied to qualitative analysis of selected complex samples in liquid and solid phase as well as an esterification reaction.

Probing Skin for Metabolites and Topical Drugs with Hydrogel Micropatches.

Sampling the skin surface is a convenient way to obtain biological specimens bearing clinically relevant information. Hydrogel micropatches enable noninvasive collection of skin excretion specimens, which can subsequently be subjected to rapid mass spectrometric analysis providing insights into the skin metabolome.

Quantitative mass spectrometry of unconventional human biological matrices.

The development of sensitive and versatile mass spectrometric methodology has fuelled interest in the analysis of metabolites and drugs in unconventional biological specimens. Here, we discuss the analysis of eight human matrices-hair, nail, breath, saliva, tears, meibum, nasal mucus and skin excretions (including sweat)-by mass spectrometry (MS). The use of such specimens brings a number of advantages, the most important being non-invasive sampling, the limited risk of adulteration and the ability to obtain information that complements blood and urine tests. The most often studied matrices are hair, breath and saliva. This review primarily focuses on endogenous (e.g. potential biomarkers, hormones) and exogenous (e.g. drugs, environmental contaminants) small molecules. The majority of analytical methods used chromatographic separation prior to MS; however, such a hyphenated methodology greatly limits analytical throughput. On the other hand, the mass spectrometric methods that exclude chromatographic separation are fast but suffer from matrix interferences. To enable development of quantitative assays for unconventional matrices, it is desirable to standardize the protocols for the analysis of each specimen and create appropriate certified reference materials. Overcoming these challenges will make analysis of unconventional human biological matrices more common in a clinical setting.This article is part of the themed issue 'Quantitative mass spectrometry'.

Hydrogel Micropatch and Mass Spectrometry-Assisted Screening for Psoriasis-Related Skin Metabolites.

BACKGROUND: Psoriasis is a chronic, immune-mediated inflammatory skin disease. Screening skin metabolites could unravel the pathophysiology of psoriasis and provide new diagnostic approaches. Due to the lack of suitable methodologies for collecting scarce amounts of skin excretions, the psoriatic skin metabolome has not been extensively studied. METHODS: We implemented biocompatible hydrogel micropatch probes combined with mass spectrometry to investigate the skin metabolome. This noninvasive approach was applied to examine samples obtained from 100 psoriatic patients and 100 healthy individuals. We also developed custom data treatment tools and used chemometric and statistical tools to reveal the alterations in the skin metabolome caused by psoriasis. RESULTS: The proposed methodology enabled us to capture alterations in the composition of skin excretions caused by the disease. Chemometric analysis revealed the major differences between the metabolomes of psoriatic skin and healthy skin. Several polar metabolites were positively (choline and glutamic acid) or negatively (urocanic acid and citrulline) correlated with the plaque severity scores. The amounts of these metabolites in the excretions sampled from psoriatic skin were significantly different (P < 0.001) from the excretions sampled from healthy skin. The role of biological variability and various confounding factors, which might affect the skin metabolome, was also investigated. CONCLUSIONS: Sampling lesional and healthy skin with the hydrogel micropatch probes and subsequent direct mass spectrometry scanning provided information on the alterations in the skin metabolome caused by psoriasis, increasing the understanding of the complex pathophysiology of this disease.

Micropatch-arrayed pads for non-invasive spatial and temporal profiling of topical drugs on skin surface.

Micropatch-arrayed pads (MAPAs) are presented as a facile and sensitive sampling method for spatial profiling of topical agents adsorbed on the surface of skin. MAPAs are 28 × 28 mm sized pieces of polytetrafluoroethylene containing plurality of cavities filled with agarose hydrogel. They are affixed onto skin for 10 min with the purpose to collect drugs applied topically. Polar compounds are absorbed by the hydrogel micropatches. The probes are subsequently scanned by an automated nanospray desorption electrospray ionization mass spectrometry system operated in the tapping dual-polarity mode. When the liquid junction gets into contact with every micropatch, polar compounds absorbed in the hydrogel matrix are desorbed and transferred to the ion source. A 3D-printed interface prevents evaporation of hydrogel micropatches assuring good reproducibility and sensitivity. MAPAs have been applied to follow dispersion of topical drugs applied to human skin in vivo and to porcine skin ex vivo, in the form of self-adhesive patches. Spatiotemporal characteristics of the drug dispersion process have been revealed using this non-invasive test. Differences between drug dispersion in vivo and ex vivo could be observed. We envision that MAPAs can be used to investigate spatiotemporal kinetics of various topical agents utilized in medical treatment.

Hydrogel micropatches for sampling and profiling skin metabolites.

Metabolites excreted by skin have a huge potential as disease biomarkers. However, due to the shortage of convenient sampling/analysis methods, the analysis of sweat has not become very popular in the clinical setting (pilocarpine iontophoresis being a prominent exception). In this report, a facile method for sampling and rapid chemical profiling of skin metabolites excreted with sweat is proposed. Metabolites released by skin (primarily the constituents of sweat) are collected into hydrogel (agarose) micropatches. Subsequently, they are extracted in an online analytical setup incorporating nanospray desorption electrospray ionization and an ion trap mass spectrometer. In a series of reference measurements, using bulk sampling and electrospray ionization mass spectrometry, various low-molecular-weight metabolites are detected in the micropatches exposed to skin. The sampling time is as short as 10 min, while the desorption time is 2 min. Technical precision of micropatch analysis varies within the range of 3-42%, depending on the sample and the method of data treatment; the best technical precision (≤10%) has been achieved while using an isotopically labeled internal standard. The limits of detection range from 7 to 278 pmol. Differences in the quantities of extracted metabolites are observed for the samples obtained from healthy individuals (intersubject variabilities: 30-89%; n = 9), which suggests that this method may have the potential to become a semiquantitative assay in clinical analysis and forensics.