Norharmane matrix enhances detection of endotoxin by MALDI-MS for simultaneous profiling of pathogen, host, and vector systems

The discovery of novel pathogenic mechanisms engaged during bacterial infections requires the evolution of advanced techniques. Here, we evaluate the dual polarity matrix norharmane (NRM) to improve detection of bacterial lipid A (endotoxin), from host and vector tissues infected with Francisella novicida (Fn). We evaluated NRM for improved detection and characterization of a wide range of lipids in both positive and negative polarities, including lipid A and phospholipids across a range of matrix assisted laser desorption-ionization (MALDI)-coupled applications. NRM matrix improved the limit of detection (LOD) for monophosphoryl lipid A (MPLA) down to picogram-level representing a ten-fold improvement of LOD versus 2,5-dihydroxybenzoic acid (DHB) and 100-fold improvement of LOD versus 9-aminoacridine (9-AA). Improved LOD for lipid A subsequently facilitated detection of the Fn lipid A major ion (m/z 1665) from extracts of infected mouse spleen and the temperature-modified Fn lipid A at m/z 1637 from infected D. variabilis ticks. Finally, we simultaneously mapped bacterial phospholipid signatures within an Fn infected spleen along with exclusively host-derived inositol-based phospholipid (m/z 933) demonstrating co-profiling for the host-pathogen interaction. Expanded use of NRM matrix in other infection models and endotoxin-targeting imaging experiments will improve our understanding of the lipid interactions at the host-pathogen interface

Changes in Monomeric and Polymeric Pigments during Chokeberry Juice Processing

Chokeberry consumption has been increasing due to research exposing great potential of health-promoting compounds. However, chokeberries are highly astringent and are typically consumed in processed forms in which product heating is applied. Processing chokeberries has been reported to degrade bioactive compounds, thus limiting the potential for consumers to obtain their health-promoting benefits. The purpose of this research was to examine the effects of chokeberry juice processing and storage on anthocyanin, flavonol, proanthocyanidin, hydroxycinamic acid, and polymeric pigment content, as well as percent polymeric color. In addition, new analytical methodologies were explored to better understand possible outcomes of polymeric pigments due to juice processing and storage. Chokeberries were processed into nonclarified juice with sampling at each stage of processing. Levels of anthocyanins, flavonols, proanthocyanidins, hydroxycinamic acids, and percent polymeric color were also analyzed each month throughout a 6 month storage period at ambient temperature. It was determined that anthocyanins readily degrade during juice processing, especially with heat applications during blanching and pasteurization. Comparatively, other compounds, such as proanthocyanidins were better retained during processing than anthocyanins, with significant levels remaining in the presscake. After pasteurization, lower levels of anthocyanins (7%), flavonols (52%), proanthocyanidins (55%), and hydroxycinamic acids (63%) remained in the juice. Alternatively, polymeric color values increased to 29% throughout processing. During juice storage, polyphenolic levels continued to decrease over 6 months while percent polymeric color values increased further to 44.5%. Little change occurred in levels of total flavonols (447.8 to 406 mg/100g), proanthocyanidins (19.7 to 16.5 mg/100g), and hydroxycinamic acids (72.7 to 48.9 mg/100g) over 6 months of storage. After observing a 55% reduction in anthocyanins due to blanching frozen chokeberries, standard juice processing was altered by removing the blanch step and its effect on polyphenolics was evaluated. The effect of two different juice processes (with and without blanching) on anthocyanin, proanthocyanidin, flavonol, and hydroxycinnamic acid contents as well as percent polymeric color was evaluated at each stage of processing. Juice pasteurization times and temperatures were also evaluated to develop a statistical model that would predict optimal anthocyanin retention. In comparison of the two processes, there were no significant differences in anthocyanin content after pasteurization; however, samples receiving no blanch had higher levels of anthocyanins after enzyme treatment. Pasteurized juice samples receiving a blanch treatment had 37% and 16% higher levels of total proanthocyanidins and flavonols, respectively than pasteurized juice receiving no blanch treatment. A response surface model was designed for the prediction of anthocyanin retention with optimum pasteurization conditions of 74°C for 2.02 minutes. MALDI-TOF-MS was used to identify large molecular weight proanthocyanidins and polymeric pigments throughout each stage of processing and over six months of juice storage. Proanthocyanidins and polymeric pigments having up to a degree of polymerization (DP) of 16 and 14, respectively, were identified in frozen berries, samples obtained throughout juice processing, and stored juices. In attempt to separate polymeric pigments, both normal phase and reverse phase TLC plates with various solvent systems were evaluated. However, only monomeric and polymeric fractions could be separated on a single plate, rather than separating each polymeric pigment by degree of polymerization. Further research is needed in order to isolate and purify polymeric pigments so that quantification methods can be developed and help explain the fate of anthocyanins during juice processing and storage

Exploring the anti‐α‐amylase activity of flavonoid aglycones in fabaceae plant extracts: a combined MALDI‐TOF‐MS and LC–MS/MS approach

A combination of TLC-bioautography, MALDI-TOF-MS and LC–MS/MS methods was used to identify flavonoids with anti-α-amylase activity in extracts of Lathyrus pratensis L. (herb), L. polyphillus L. (fruits), Thermopsis lanceolata R. Br. (herb) and S. japonica L. (buds). After the TLC-autobiography assay, substances with anti-amylase activity were identified by MALDI-TOF-MS followed by confirmation of the result by LC–MS/MS. Results of the study revealed that the flavonoids apigenin, luteolin, formononetin, genistein and kaempferol display marked anti-α-amylase activity. Formononetin showed the largest activity. Compared with LC–MS/MS, MALDI-TOF-MS is a quick and convenient method; results can be obtained within minutes; and only minor sample amounts are required which allows us to analyse mixtures of substances without preliminary separation. However, the inability to distinguish between isomers is the main limitation of the method.BAM/BMWi http://dx.doi.org/10.13039/501100002765German Research Foundation http://dx.doi.org/10.13039/501100001659Peer Reviewe

Thin layer chromatography-matrix assisted laser desorption ionisation-mass spectrometry of pharmaceutical compounds.

Thin-layer chromatography (TLC) is of great importance for the pharmaceutical industry as a simple, quick, and low cost analytical method. Considerable effort has been made over the past decades to combine the simplicity of TLC with the selectivity and sensitivity of mass spectrometry (MS) detection. In the pharmaceutical industry sensitivity is an especially important factor, since the allowed impurity level of most drugs is under 0.1%.The aim of the present thesis was to develop methods for the direct examination of pharmaceutical compounds from TLC plates by matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry (MALDI TOF MS). The study was started by comparing several approaches for the application of the matrix for direct TLC-MALDI including a newly developed electrospray matrix deposition method. This new method was found to be superior to the other techniques studied. It produced a stable signal, minimised analyte spreading, and hence allowed the scanning of a TLC plate to obtain chromatographic as well as mass spectral data. The plotted mass chromatograms assisted in spot location, and allowed the calculation of Rf-values. These showed good agreement with the Rf -values determined by UV detection. The decrease in mass resolution and mass accuracy commonly observed in TLC-MALDI TOF MS due to the uneven nature of the silica gel layer was corrected by internal recalibration on selected matrix ions during the scanning of the TLC plate. To enhance the signals recorded directly from a TLC plate the use of an extraction solvent prior the matrix application was explored. Further improvements in sensitivity were obtained by modifying a robotic x-y-z axis motion system to act as an electrospray deposition device and by use of special Si 60 F[254] HPTLC-MALDI targets. Using both approaches sensitivities in the high fmol range were obtained. To minimise matrix interference, which can suppress analyte signals, the application of suspensions of particles of different materials and sizes (Co-UFP, TiN, TiO[2], graphite and silicon) onto eluted TLC plates were investigated. The structural analysis of pharmaceutical compounds was achieved by post-source decay - matrix-assisted laser desorption/ionisation (PSD-MALDI) mass spectrometry performed directly on the separated spots. TLC-MALDI MS is not only applicable to the qualitative analysis of pharmaceutical compounds. The generation of quantitative data by using a structural analogue as an internal standard is also described. Different approaches to the incorporation of the internal standard into the TLC plate were tested. The most successful approach was to develop the TLC plate in the mobile phase to which the internal standard was added. Good accuracy, precision, linearity and sensitivity was obtained using this approach

Distribution of anti-cancer drugs in solid tumours studied by MALDI-MSI.

Vascular disrupting agents (VDAs) have been used in treatment of many cancers. 5,6 -dimethylxanthenone-4-acetic acid (DMXAA) is a low molecular weight drug of the flavonoid group which has an anti-vascular effect in tumours causing endothelial cell apoptosis and activation of cytokines.A study employing matrix assisted laser desorption ionisation-mass spectrometry (MALDI-MS) imaging to examine LS174T colorectal adenocarcinoma xenografts following administration of DMXAA has been conducted to study the distribution of anti-cancer drugs and to explore markers of efficacy and resistance. Initial work established the limit of detection /quantitation of DMXAA in tissue. The drug limit of detection (LoD) is determined as 10 ng/ml and the drug lower limit of quantitation (LLoQ) is 45 ng/ml. MALDI images were recorded from LS174T colorectal adenocarcinoma xenografts removed from immunodeficient mice following treatment with with 27.5 mg/kg DMXAA. These indicated that the drug was distributed mainly in the centre of tumour 4h post-treatment, whilst it was distributed around the periphery 24h posttreatment.A study of lipid expression in treated tumors demonstrated that washing tissue sections with 150 mM ammonium acetate solution (NH[4]AC) improved the intensity of lipids signals in both negative and positive ion mode. These images also indicated that sphingomyelins (SM) and phosphatidylcholines (PC) lipid species were highly expressed in cancerous tissue.A thin layer chromatography-matrix assisted laser desorption ionisation-mass spectrometry (TLC-MALDI-MS) experiment has been carried out for the analysis of phospholipids extracted from the treated xenograft tumours. The lipid extracts were separated into 6 spots on the TLC plate. These were identified as lysophosphatidylcholines (LPC), sphingomyelins (SM), phosphatidylcholines (PC) and phosphatidylethanolamines (PE). The TLC-MALDI-MS data indicated that LPC were highly expressed in the 4h and 24h post-treated tumour samples compared to the control. An increase in expression of LPC lipids in solid tumours treated with DMXAA has been demonstrated and shown to be localised in the central area of the tumour.Mass spectrometry imaging was also used to characterise proteins and peptide signal in tumours after treatment with DMXAA. Histone H2A peaks at 944 m/z were highly expressed in the region of the tumours. In addition, a characteristic increase in the Hb beta chain at 1274.74 m/z in the 24h post-treated tumour has been seen. The data obtained from PCA has shown that the levels of certain proteins changed over the different tumour time point

Thin-Layer Chromatography with Ultraviolet and Mass Spectrometric Detection : From Preparative-Layer to Miniaturized Ultra-Thin-Layer Technique

The present challenge in drug discovery is to synthesize new compounds efficiently in minimal time. The trend is towards carefully designed and well-characterized compound libraries because fast and effective synthesis methods easily produce thousands of new compounds. The need for rapid and reliable analysis methods is increased at the same time. Quality assessment, including the identification and purity tests, is highly important since false (negative or positive) results, for instance in tests of biological activity or determination of early-ADME parameters in vitro (the pharmacokinetic study of drug absorption, distribution, metabolism, and excretion), must be avoided. This thesis summarizes the principles of classical planar chromatographic separation combined with ultraviolet (UV) and mass spectrometric (MS) detection, and introduces powerful, rapid, easy, low-cost, and alternative tools and techniques for qualitative and quantitative analysis of small drug or drug-like molecules. High performance thin-layer chromatography (HPTLC) was introduced and evaluated for fast semi-quantitative assessment of the purity of synthesis target compounds. HPTLC methods were compared with the liquid chromatography (LC) methods. Electrospray ionization mass spectrometry (ESI MS) and atmospheric pressure matrix-assisted laser desorption/ionization MS (AP MALDI MS) were used to identify and confirm the product zones on the plate. AP MALDI MS was rapid, and easy to carry out directly on the plate without scraping. The PLC method was used to isolate target compounds from crude synthesized products and purify them for bioactivity and preliminary ADME tests. Ultra-thin-layer chromatography (UTLC) with AP MALDI MS and desorption electrospray ionization mass spectrometry (DESI MS) was introduced and studied for the first time. Because of the thinner adsorbent layer, the monolithic UTLC plate provided 10 100 times better sensitivity in MALDI analysis than did HPTLC plates. The limits of detection (LODs) down to low picomole range were demonstrated for UTLC AP MALDI and UTLC DESI MS. In a comparison of AP and vacuum MALDI MS detection for UTLC plates, desorption from the irregular surface of the plates with the combination of an external AP MALDI ion source and an ion trap instrument provided clearly less variation in mass accuracy than the vacuum MALDI time-of-flight (TOF) instrument. The performance of the two-dimensional (2D) UTLC separation with AP MALDI MS method was studied for the first time. The influence of the urine matrix on the separation and the repeatability was evaluated with benzodiazepines as model substances in human urine. The applicability of 2D UTLC AP MALDI MS was demonstrated in the detection of metabolites in an authentic urine sample.Uusia lääkeaineita on perinteisesti etsitty luonnosta mutta luonnosta saatavien yhdisteiden kirjo ei ole kuitenkaan kyennyt kattamaan nykypäivän lääkkeenkehityksen tarvetta. Haasteena on saada markkinoille uusia yhdisteitä jo myynnissä olevien lääkeaineiden rinnalle. Tavoitteena on syntetisoida uusia yhdisteitä lääkeainekandidaateiksi mahdollisimman tehokkaasti. Näiden uusien yhdisteiden biologinen aktiivisuus ja ADME-ominaisuudet (lääkeaineen imeytyminen, jakautuminen, metabolia, ja eritys) testataan elimistön ulkopuolella jo paljon ennen ihmisillä tehtäviä kliinisiä kokeita, jotta lääkkeiksi kelvottomat yhdisteet voidaan seuloa pois mahdollisimman varhaisessa vaiheessa. Uusien yhdisteiden tuottamisessa käytettävät synteesimenetelmät ovat tehokkaita ja nopeita, ja siksi myös uusien yhdistekirjastojen testausmenetelmien on oltava nopeita laadun varmistamiseksi. Nopeat, yksinkertaiset, taloudelliset ja herkät testausmenetelmät mahdollistavat laajojen yhdistekirjastojen nopean ja tehokkaan puhtausseulonnan ja luovat pohjan kirjastojen optimaaliseen laadunvalvontaan. Tällöin varhais-ADME tutkimuksissa ja biologisen aktiivisuuden seulonnassa vältytään vääriltä positiivisilta tai negatiivisilta tuloksilta. Tässä väitöskirjatyössä tutkittiin ohutkerroskromatografian ja ultravioletti ja massaspektrometristen havainnointimenetelmien soveltuvuutta oikeellisuus- ja puhtaustutkimukseen. Työssä analysoitiin jo markkinoilla olevia lääkeaineita, synteesikirjaston yhdisteitä (lääkeainekandidaatteja) ja biologisia näytteitä. Yhdisteiden puhdistamisessa käytettiin preparatiivista tasokromatografiaa (PLC) ja laadunvarmistukseen liittyvissä tutkimuksissa korkean erotuskyvyn ohutkerroskromatografiaa (HPTLC) ja ultraohutkerroskromatografiaa (UTLC). Ohutkerroskromatografisilla menetelmillä saatuja tuloksia verrattiin myös nestekromatografialla saatuhin tuloksiin, koska nestekromatografia on valtamenetelmä nykypäivän laadunvarmistustesteissä. Massaspektrometrisista menetelmistä käytettiin matriisiavusteista laserdesorptio/ionisaatiomassaspektrometriaa (MALDI), ja sähkösumutus- (ESI) ja desorptiosähkösumutusionisaatio (DESI) massaspektrometriaa. Työn tuloksena kehitettiin käyttökelpoisia, nopeita ja tehokkaita menetelmiä pienten lääkeaine- ja lääkeainekandidaattimolekyylien laadulliseen ja määrälliseen tutkimiseen

New Applications of Mass Spectrometry for Drug and Lipid Analysis

Mass spectrometry is an important tool used in many different disciplines and settings that include forensics, drug discovery, environmental analysis, and proteomics. Gas chromatography - mass spectrometry (GC-MS) and matrix assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI - TOF MS) are two of the most important instruments used for analysis of compounds. Chapters 1 and 2 of this discussion use GC-MS for the investigation of synthetic cannabinoids in `K2\u27 incense products and the detection of metabolites in urine samples from individuals suspected of consuming these mixtures. Analytical standards were synthesized and used for identification and confirmation of structures. Detection of these compounds and their metabolites is important since `K2\u27 products are banned nationwide as a Schedule 1 substance. Chapter 3 uses LDI-TOF MS for the analysis of triacylglycerol (TAG) degradation products in fingermark samples exposed to light/dark conditions on four different surfaces: stainless steel, glass, plastic, and iron. A standard of triolein was used for identification of products with the detection of C7:0 and C8:0 aldehyde and carboxylic acids through tandem mass spectrometry analysis. The age of a fingermark was estimated through comparison of unsaturated and saturated TAGs. Analysis of TAGs in fingermarks is important as a possible dating technique which could provide investigative leads or a timeline of events in a criminal investigation. Chapter 4 uses MALDI-TOF MS for the development of a rapid separation technique to overcome suppression effects of TAGs by phosphatidylcholine (PC). A solid phase extraction (SPE) technique was developed to separate these classes of compounds using reference lipids and real samples (beef, egg yolk) as models. Because lipids play important roles in biological systems, a method to clearly detect and overcome suppression effects is important. Chapter 5 uses GC-MS for the analysis of fatty and resin acids in biomass fermentation process waters. An extraction procedure was developed for detection and quantification of these compounds. Monitoring these acids in water samples is important because they can negatively impact environmental and industrial pathways