Combination Of Thin-layer Chromatography Research Articles

Rapid Raman spectroscopic identification of three homoisoflavanones in polygonatum odoratum based on twice chromatography derivatization

The aim of this study was to establish a new method for the determination of homoisoflavanones (Ⅲ, Ⅳ, V) in polygonatum odoratum(POD) by combination of thin layer chromatography (TLC) and chemical derivative resonance Raman spectroscopy (RRS). The twice chromatography method of TLC was used to improve the specificity of the component to be tested; the method of the relative Rf was used to reduce the use of the reference substance of the component to be tested; the chemical derivatization method was used to improve the signal intensity of Raman spectrum for the component to be tested in POD, so as to obtain a trace amount fingerprint structure information of the measured component. The method exhibits robust specificity, high sensitivity, and reliable stability, there by offering a novel reference approach for the identification and evaluation of homoisoflavanones (Ⅲ, Ⅳ, V) in POD.

Development of a Thin-Layer Chromatography-Enzymatic Test Combination Method for the Isolation of α-Glucosidase Inhibitors From Thymelaea hirsuta.

A rapid, easy and simple method for the isolation and purification of α-glucosidase inhibitors of the ethyl acetate extract of Thymelaea hirsuta (EaTh) by a combination of thin layer chromatography (TLC) and enzymatic test has been developed. EaTh was demonstrated previously a potent α-glucosidase inhibitory effect. In this study, we developed a simple TLC-enzymatic test (TLC/EZ) combination to isolate α-glucosidase inhibitors present in EaTh.EaTh was extracted by Soxhlet from Thymelaea hirsuta (T. hirsuta). The EaTh was separated on a silica gel column and then on a TLC plate. After TLC separation, the TLC/EZ combination method was applied. α-glucosidase inhibitors were detected directly in the TLC plate using the glucose oxidase peroxidase method (GOD-POD). A good detection of active compounds was obtained in the TLC favoring the TLC/EZ method. Active compounds were then characterized using high performance liquid chromatography-mass spectrometry (HPLC-MS) analysis. The main α-glucosidase inhibitors present in EaTh have a molecular ion [M + H]+ at m/z = 543. This proposed method is suitable for a reliable isolation and purification of α-glucosidase inhibitors present in EaTh. It could be proposed as an interesting alternative of the classical method for the isolation and purification of α-glucosidase inhibitors in plant extracts.

High-performance thin-layer chromatography in combination with an acetylcholinesterase-inhibition bioassay with pre-oxidation of organothiophosphates to determine neurotoxic effects in storm, waste, and surface water

Pesticides such as organothiophosphates (OTPs) are neurotoxically active and enter the aquatic environment. Bioassays, using acetylcholinesterase (AChE), a suitable substrate and reactant, can be applied for the photometric detection of AChE-inhibiton (AChE-I) effects. The oxidized forms of OTPs, so-called oxons, have higher inhibition potentials for AChE. Therefore, a higher sensitivity is achieved for application of oxidized samples to the AChE assay. In this study, the oxidation of malathion, parathion, and chlorpyrifos by n-bromosuccinimide (NBS) was investigated in an approach combining high-performance thin-layer chromatography (HPTLC) with an AChE-I assay. Two AChE application approaches, immersion and spraying, were compared regarding sensitivity, precision, and general feasibility of the OTP effect detection. The oxidation by NBS led to an activation of the OTPs and a strong increase in sensitivity similar to the oxons tested. The sensitivity and precision of the two application techniques were similar, although the spray method was slightly more sensitive to the oxidized OTPs. The 10% inhibition concentrations (IC10) for the spray approach were 0.26, 0.75, and 0.35 ng/spot for activated malathion, parathion, and chlorpyrifos, respectively. AChE-I effect recoveries in samples from a stormwater retention basin and receiving stream were between 69 and 92% for malathion, parathion, and chlorpyrifos. The overall workflow, including sample enrichment by solid-phase extraction, HPTLC, oxidation of OTPs, and AChE-I assay, was demonstrated to be suitable for the detection of AChE-I effects in native water samples. An effect of unknown origin was found in a sample from a stormwater retention basin.

Influence of Chromatographic Conditions on LOD and LOQ of Fluoxetine and Sertraline Analyzed by TLC-Densitometric Method

This research introduces the analysis of fluoxetine and sertraline by means of the TLC-densitometric method. They provide information on LOD and LOQ under various chromatographic conditions. The study used adsorption (NPTLC) and partition (RPTLC) thin-layer chromatography in combination with a densitometric analysis. Four types of chromatographic plates precoated with: silica gel 60 F254, silica gel 60, silanized silica gel 60 F254 (RP-2), and a mixture of silica gel 60 and kieselguhr F254, as well as three mobile phases: chloroform + methanol + ammonia (9:1:0.4, v/v/v), chloroform + methanol + glacial acetic acid (5:4:1, v/v/v), and acetone + toluene + ammonia (10:9:1, v/v/v), were used in NPTLC. RP-18F254 and silanized silica gel 60 F254 (RP-2) plates and four mobile phases: methanol + water (10:0 and 9:1, v/v), acetone + water (10:0 and 9:1, v/v), were used in RPTLC. The lowest LOD and LOQ values for fluoxetine were obtained using a silanized silica gel 60 F254 (RP-2) with acetone + toluene + ammonia (10:9:1, v/v/v) in NPTLC, and with a silanized silica gel 60 F254 (RP-2) in combination with methanol + water (10:0, v/v) in RPTLC. The lowest LOD and LOQ values of sertraline were obtained using a silica gel 60 with acetone + toluene + ammonia (10:9:1; v/v/v) in NPTLC. The smallest amount of sertraline was detected on the silanized silica gel 60 F254 plate in combination with methanol + water (9:1, v/v) in RPTLC. The obtained results provide important information that can give a good basis and set the direction for further, more detailed research; the results can also benefit other researchers who analyze fluoxetine and sertraline with the TLC technique in model systems (testing standards) as well as in drug and biological samples.

Recent Advances in Combinations of TLC With MALDI and Other Desorption/Ionization Mass-Spectrometry Techniques.

The combination of planar chromatography with desorption/ionization mass-spectrometry (MS) techniques provides chemists with unique tools for fast and simple separation of mixtures followed by the detection of analytes by the most powerful analytical method. Since its introduction in the early 1990s, thin-layer chromatography (TLC)/matrix-assisted mass spectrometry (MALDI) has been used for the analysis of a wide range of analytes, including natural and synthetic organic compounds. Nowadays, new desorption/ionization approaches have been developed and applied in conjunction with planar chromatography competing with MALDI. This review covers recent developments in the combination of TLC with various desorption/ionization MS methods which were made in recent several years.

Elucidating the degradation pattern of a new cold-tolerant pectate lyase used for efficient preparation of pectin oligosaccharides

The cold-active pectate lyases have drawn increasing attention in food and biotechnological applications due to their ability to retain high catalytic efficiency under lower temperatures, which could be helpful for energy saving, cost reduction and flavor preservation. Herein, a new cold-tolerant pectate lyase (ErPelPL1) gene from Echinicola rosea was cloned and heterologously expressed in Escherichia coli. Interestingly, ErPelPL1 retained high catalytic activity even at a low temperature (4 °C). ErPelPL1 exhibited optimal activity at 35 ℃, pH 8.0 with 1 mM of Ca2+. It showed high specific activity towards polygalacturonic acid (34.7 U/mg) and sodium polygalacturonate (59.3 U/mg). The combined thin-layer chromatography (TLC), fast protein liquid chromatography (FPLC) and electrospray ionization mass spectrometry (ESI-MS) results indicated that ErPelPL1 endolytically degraded pectic substances into the oligosaccharides with degrees of depolymerization (Dps) of 1–6. In conclusion, this study mainly conducted biochemical characterization and product analysis of a cold-tolerant pectate lyase. Therefore, it provides a promising enzyme candidate for food and biotechnological applications.Graphical

High-performance thin-layer chromatography in combination with a yeast-based multi-effect bioassay to determine endocrine effects in environmental samples.

Effect-directed analysis (EDA) that combines effect-based methods (EBMs) with high-performance thin-layer chromatography (HPTLC) is a useful technique for spatial, temporal, and process-related effect evaluation and may provide a link between effect testing and responsible substance identification. In this study, a yeast multi endocrine-effect screen (YMEES) for the detection of endocrine effects is combined with HPTLC. Simultaneous detection of estrogenic, androgenic, and gestagenic effects on the HPTLC plate is achieved by mixing different genetically modified Arxula adeninivorans yeast strains, which contain either the human estrogen, androgen, or progesterone receptor. Depending on the yeast strain, different fluorescent proteins are formed when an appropriate substance binds to the specific hormone receptor. This allows to measure hormonal effects at different wavelengths. Two yeast cell application approaches, immersion and spraying, are compared. The sensitivity and reproducibility of the method are shown by dose-response investigations for reference compounds. The spraying approach indicated similar sensitivities and higher precisions for the tested hormones compared to immersion. The EC10s for estrone (E1), 17β-estradiol (E2), 17α-ethinylestradiol (EE2), 5α-dihydrotestosterone (DHT), and progesterone (P4) were 95, 1.4, 10, 7.4, and 15pg/spot, respectively. Recovery rates of E1, E2, EE2, DHT, and P4 between 88 and 120% show the usability of the general method in combination with sample enrichment by solid phase extraction (SPE). The simultaneous detection of estrogenic, androgenic, and gestagenic effects in wastewater and surface water samples demonstrates the successful application of the YMEES in such matrices. This promising method allows us to identify more than one endocrine effect on the same HPTLC plate, which saves time and material. The method could be used for comparison, evaluation, and monitoring of different river sites and wastewater treatment steps and should be tested in further studies.

Characterization of glycosphingolipids from gastrointestinal stromal tumours

Gastrointestinal stromal tumours (GISTs) are the major nonepithelial neoplasms of the human gastrointestinal tract with a worldwide incidence between 11 and 15 per million cases annually. In this study the acid and non-acid glycosphingolipids of three GISTs were characterized using a combination of thin-layer chromatography, chemical staining, binding of carbohydrate recognizing ligands, and mass spectrometry. In the non-acid glycosphingolipid fractions of the tumors globotetraosylceramide, neolactotetraosylceramide, and glycosphingolipids with terminal blood group A, B, H, Lex, Lea, Ley and Leb determinants were found. The relative amounts of these non-acid compounds were different in the three tumour samples. The acid glycosphingolipid fractions had sulfatide, and the gangliosides GM3, GD3, GM1, Neu5Acα3neolactotetraosylceramide, GD1a, GT1b and GQ1b. In summary, we have characterized the glycosphingolipids of GISTs and found that the pattern differs in tumours from different individuals. This detailed characterization of glycosphingolipid composition of GISTs could contribute to recognition of new molecular targets for GIST treatment and sub-classification.

Utilization of a Crown Ether/Amine-Type Rotaxane as a Probe for the Versatile Detection of Anions and Acids by Thin-Layer Chromatography.

A crown ether/amine-type [2]rotaxane was synthesized and utilized as a probe for the detection of acids and anions. The addition of acids to the amine-type [2]rotaxane solution generated corresponding crown ether/ammonium-type [2]rotaxanes, which were purified by silica gel column chromatography as ammonium salts. The isolated yields of the [2]rotaxanes, possessing a variety of anions, depended on the acidity and polarity of the counter anions. The behaviours of the ammonium-type [2]rotaxanes on thin-layer chromatography (TLC) silica gel reflected the properties of the counter anions. The treatment of the amine-type [2]rotaxane with acids afforded the corresponding ammonium-type [2]rotaxanes bearing several different anions. The ammonium-type [2]rotaxanes behaved similarly to the purified [2]rotaxanes on the TLC silica gel. Furthermore, we succeeded in the analysis of anions using mixtures of the amine-type [2]rotaxane and salts in an appropriate solvent. We demonstrated the detection of anions by the combination of TLC and the utilization of the [2]rotaxane probe.

Estimating Limits of Detection and Quantification of Ibuprofen by TLC-Densitometry at Different Chromatographic Conditions

Ibuprofen is one of the best-known nonsteroidal anti-inflammatory and analgesic drugs. Following the previous work, the current study is focused on estimating the effect of different chromatographic conditions on the sensitivity of thin-layer chromatography in combination with UV densitometry, i.e., the detection and quantification of ibuprofen in a wide range of its concentrations including the lowest limits of detection (LOD) and quantification (LOQ). For this purpose, a reliable and easy-to-use calculation procedure for LOD and LOQ determination is presented in this work. In addition, the impact of type plates and mobile phase composition on the LOD and LOQ, respectively, of this active substance is accurately described. The results of detection and the quantification level of ibuprofen obtained under applied chromatographic conditions confirmed the utility of silica gel plates as well as silica gel bonded phases (i.e., reversed-phase (RP) plates) in the thin-layer chromatography (TLC)-densitometric analysis of ibuprofen at a low level, i.e., from several nanograms (below one microgram) to a few micrograms/spot. Among all chromatographic systems tested, the best are those consisting of silica gel 60F254 plates with concentrating zone (1.05583) and the mixture of n-hexane:ethyl acetate:glacial acetic acid in ratio 36:12:2 (v/v/v) as well as RP plates, i.e., RP-18F254 aluminum plates developed with methanol:water in volume composition 40:10 (v/v). These chromatographic systems allowed quantifying ibuprofen in the amount of 0.229 µg/spot and 0.228 µg/spot, which is less than 1 µg/spot. It can be stated that different chromatographic systems in combination with UV densitometric scanning at 224 nm proposed in this work can be successfully applied for the cost-effective and sensitive determination of ibuprofen as a widely used drug component as well as a residual in domestic wastewater. It was found that the modification of silica gel as well as the layer thickness of unmodified or modified silica gel 60 can influence the quality of chromatograms and the detection/quantification of ibuprofen in both normal phase (NP) and RP systems. Therefore, to obtain the best possible LOD and LOQ values of ibuprofen with precoated layers, suitable mobile phase and chromatographic plates are required.

High-performance thin-layer chromatography hyphenated to high-performance liquid chromatography-diode array detection-mass spectrometry for characterization of coeluting isomers

The effect-directed analysis on a planar chromatogram allows for fast non-target screening, multi-imaging detection of effects (bioprofiling) and highly targeted characterization and isolation of bioactive compounds. For direct characterization by high-resolution mass spectrometry (HRMS), however, the orthogonal hyphenation of two different liquid chromatographic techniques (planar and column chromatography) is still underexplored. In particular, it can be helpful in case of coeluting compounds. Exemplarily, lemon balm (Melissa officinalis L.) leaf extract was analysed by high-performance thin-layer chromatography in combination with bioactivity assays for antibacterial (against the Gram-positive Bacillus subtilis and the Gram-negative Aliivibrio fischeri) and α-glucosidase-inhibitory compounds (HPTLC-UV/Vis/FLD-EDA). High-resolution mass spectra of two bioactive compound zones were directly recorded via an elution head-based interface. By HPTLC-HESI-HRMS, the compound in zone a inhibited A. fischeri and was identified as linolenic acid, whereas the two closely related constitutional isomers oleanolic acid and ursolic acid were present in zone b. This was proven by two-dimensional liquid chromatography. Heart-cutting HPTLC-UV/Vis/FLD-HPLC-DAD-MS allowed the separation of the two isomers and proved both to be present in the bioactive zone with ursolic acid at a much higher abundance.

Combination of Thin-Layer Chromatography and Mass Spectrometry Using Cluster-Induced Desorption/Ionization.

Desorption/ionization induced by neutral clusters (DINeC) was employed for mass spectrometry (MS) of oligopeptides and lipids after separation by means of thin-layer chromatography (TLC). Clear and fragmentation-free spectra were obtained from the TLC plates without any further sample treatment. Mass-resolved chromatograms were deduced when scanning the TLC plates with the cluster beam along the direction of solvent movement. Using vancomycin and noncovalently bound complexes, the soft nature of DINeC was demonstrated also when used in combination with TLC. As a test application, TLC and DINeC–MS were employed to separate and detect different phospholipids obtained from egg yolk.

Application of Thin-Layer Chromatography in Combination with Densitometry for the Determination of Diclofenac in Enteric Coated Tablets.

Diclofenac belongs to the drug class non-steroidal anti-inflammatory drugs widely used in Europe as well as all over the world. Thus, it is important to conduct research on its quality control of available pharmaceutical preparations like for example enteric coated tablets. Among various analytical techniques, thin-layer chromatography (TLC) is ideal for this task due to their short time analysis, ease of operation and low cost. Hence, the aim of this study was to develop the optimal conditions of analysis and quantitative determination of diclofenac sodium in enteric tablets by using TLC in combination with densitometry. Of all chromatographic systems tested, the best is the one which consists of silica gel 60F254 and cyclohexane: chloroform:methanol:glacial acetic acid (6:3:0.5:0.5 v/v) as the mobile phase, which allows the successful separation of examined diclofenac sodium as active component and the largest number (twelve) of its degradation products as potential impurities of its pharmaceutical products. This indicates that the newly developed method is more effective than previously reported assays by Starek and Krzek. Linearity range was found to be 4.00–18.00 μg/spot for diclofenac sodium. The results of the assay of enteric tablet formulations equals 98.8% of diclofenac sodium in relation to label claim is in a good agreement with pharmaceutical requirements.

The antifungal and Cryptococcus neoformans virulence attenuating activity of Pelargonium sidoides extracts

Ethnopharmacological relevanceLimitations of clinical antifungal treatments and drug-resistance are drivers of the search for novel antifungal strategies. Extracts prepared from the tubers of the medicinal plant, Pelargonium sidoides, are known for their antiviral and antibacterial activities and are used in ethnomedicine for the treatment of acute respiratory infections. Their impact on fungi has not been well characterised. Here, we provide a first report on the antifungal activity of a P. sidoides aerial tissue extract against Cryptococcus neoformans as well as the effects of both tuber and aerial tissue extracts on selected virulence factors. Aim of the studyNovel antimicrobial strategies that target multiple cellular pathways or make use of anti-pathogenic compounds that inhibit virulence factors have been proposed. This work aimed to evaluate P. sidoides plant parts for their anticryptococcal activity and antipathogenic properties on selected virulence factors. Materials and methodsThe antifungal activity of crude P. sidoides tuber and aerial tissue extracts (15% m/m ethanol) were compared using a modified colourimetric antifungal susceptibility test. Fungicidal activity of the extracts was confirmed by plate counts. To test yeast resistance to the extracts, it was conditioned by multiple passages in sub-lethal doses followed by antifungal susceptibility testing. Cytotoxicity of the extracts was tested with a blood agar haemolysis assay. Extracts were evaluated for the presence of multiple bioactive compounds by solid-phase fractionation and visualisation by thin-layer chromatography in combination with bioassays. The influence of extracts on the production of the polysaccharide capsule, ergosterol content as well as laccase and urease activities were also evaluated. Cell surface variations after extract exposure were visualised by scanning electron microscopy (SEM). ResultsBoth tuber and aerial tissue extracts were fungicidal and contained multiple bioactive compounds which constrained the development of antifungal resistance. No haemolytic activity was observed, and the extracts did not appear to target ergosterol biosynthesis. However, the extracts displayed anti-pathogenic potential by significantly inhibiting laccase and urease activity while also significantly reducing capsule size. SEM revealed notable cell surface variations and provided support for the observed reduction in capsule size. ConclusionsOur results provide support to the exploration of medicinal plants as sources of alternative antifungal therapies and the potential use of multicomponent inhibition and or virulence attenuation for next-generation treatment strategies. Our data also provide relevant information that may support the further use of P. sidoides in traditional medicines as well as in commercialised phytopharmaceuticals.

A New Post-Chromatographic Derivatization Approach to the Identification of Alcohols and Phenols in Complex Mixtures by a Combination of Planar Chromatography and Matrix-Assisted Laser Desorption/Ionization Mass Spectrometry

A simple and suitable approach to the post-chromatographic derivatization in the analysis of alcohol and phenols mixtures by a combination of thin-layer chromatography and MALDI mass spectrometry was proposed for the first time. The chemical modification of analytes was accomplished by the treatment of eluted zones on thin-layer chromatograms by 3-bromopropionyl chloride in the presence of an excess of a base (pyridine or triethylamine). The resulting derivatives contained a residue with a constant charge (ammonium fragment) that ensures the efficient desorption of the derivatized analytes from the sorbent layer during MALDI. It was shown that the proposed approach allows the recording of mass spectra with a high signal/noise ratio and the detection of alcohols and phenols of different structures. The reproducibility of such MALDI mass spectra is rather high, which predetermines a possibility of building chromatogram curves using total or selected ion current and, hence, their visualization and treatment on the quantitative level.

A Screening Method for the Isolation of Bacteria Capable of Degrading Toxic Steroidal Glycoalkaloids Present in Potato.

Potato juice, a by-product of starch processing, is a potential high-value food ingredient due to its high protein content. However, conversion from feed to human protein requires the removal of the toxic antinutritional glycoalkaloids (GAs) α-chaconine and α-solanine. Detoxification by enzymatic removal could potentially provide an effective and environmentally friendly process for potato-derived food protein production. While degradation of GAs by microorganisms has been documented, there exists limited knowledge on the enzymes involved and in particular how bacteria degrade and metabolize GAs. Here we describe a series of methods for the isolation, screening, and selection of GA-degrading bacteria. Bacterial cultures from soils surrounding greened potatoes, including the potato peels, were established and select bacterial isolates were studied. Screening of bacterial crude extracts for the ability to hydrolyze GAs was performed using a combination of thin layer chromatography (TLC), high performance liquid chromatography (HPLC), and liquid chromatography mass spectrometry (LC-MS). Analysis of the 16S rRNA sequences revealed that bacteria within the genus Arthrobacter were among the most efficient GA-degrading strains.

Classification of Romanian medicinal plant extracts according to the therapeutic effects using thin layer chromatography and robust chemometrics

Thin layer chromatography in combination with image analysis and advanced chemometric methods were successfully used to classify the medicinal herbs according to their therapeutic effects and usage. The investigations were conducted using two types of plates (HPTLC Silica gel 60 and HPTLC Silica gel 60 F254) which were evaluated in UV light at 254 and 365 nm. The holistic evaluation of the numerical data corresponding different image processing channels (blue, grey, red, green) was performed by employing appropriate multivariate methods: hierarchical cluster analysis (HCA), principal component analysis (PCA), fuzzy principal component analysis (FPCA) and linear discriminant analysis (LDA) applied to the first relevant principal components. The results obtained by applying LDA method indicate a highly accurate separation of the medicinal herbs within the four groups, in good agreement with therapeutic effects and usage. According to this classification, the best image processing channels were identified for each of the investigated HPTLC plates: blue channel for HPTLC Silica gel 60 F254 (with 92.9% percent of discrimination in case of PCA and FPCA) and respectively red channel for HPTLC Silica gel 60 (with 93.9% percent of discrimination in case of FPCA). The 2D and 3D score scatterplots illustrate also the accurate and reliable discrimination between the four distinct groups.

Diverse Odd-Chain Monoenoic Fatty Acids and Novel Non-Methylene-Interrupted Heptadecadienoic Acids in Ovaries of the Limpet Cellana toreuma.

Highly diverse dienoic fatty acids (FA), especially non-methylene-interrupted (NMI) FA, have been recognized in triacylglycerols (TAG) and polar lipids of ovaries of the limpet Cellana toreuma. To gain additional insights into the structural diversity of FA in ovaries, this study aimed to clarify previously undescribed minor monoenoic FA isomers and unidentified minor NMI FA by combined argentation thin-layer chromatography (TLC) and gas chromatography-mass spectrometry (GC-MS) of their 3-pyridylcarbinol derivatives. A total 66 monoenoic FA from C14 to C22, including 12 branched-chain FA, were identified. A surprising finding was a series of 13 heptadecenoic acids with double bonds at either Δ5, Δ6, Δ7, Δ8, Δ9, Δ10, Δ11, Δ12, Δ13, Δ15, or Δ16 positions. Besides previously identified NMI FA, 7,18-nonadecadienoic (19:2Δ7,18), 9,18-nonadecadienoic (19:2Δ9,18), 11,18-nonadecadienoic (19:2Δ11,18), 7,20-heneicosadienoic (21:2Δ7,20), and 11,20-heneicosadienoic (21:2Δ11,20) acids, two novel related NMI FA, 5,16-heptadecadienoic (17:2Δ5,16) and 7,16-heptadecadienoic (17:2Δ7,16) acids, were found in TAG. These findings could provide new insights into clarifying the biological characteristics of marine NMI FA.

Post-chromatographic fixed-charge derivatization for the analysis of hydroxyl-containing compounds by a combination of thin-layer chromatography and matrix-assisted laser desorption/ionization mass spectrometry

A simple and convenient on-spot derivatization has been suggested for the modification of hydroxyl-containing compounds for their analysis by thin layer chromatography/matrix-assisted laser desorption ionization mass spectrometry (TLC/MALDI). The proposed approach was based on post-chromatographic acylation of separated analytes by 3-bromopropionyl chloride with simultaneous quaternization of pyridine. In contrast to the initial alcohols not ionizable in TLC/MALDI conditions, the derivatives, containing permanent positive charge, revealintense peaks of their cationic moieties in MALDI mass spectra recorded directly from TLC plates. The method was tested on a series of mammalian and plant sterols, phenols and terpene alcohols.

Discovery and Characterization of a Novel Chitosanase from Paenibacillus dendritiformis by Phylogeny-Based Enzymatic Product Specificity Prediction.

In the process of genome mining for novel chitosanases by phylogeny-based enzymatic product specificity prediction, a gene named Csn-PD from Paenibacillus dendritiformis was discovered. The enzyme was classified as a member of the GH46 family of glycoside hydrolase based on sequence alignment, and it was functionally expressed in Escherichia coli BL21 (DE3). The recombinant chitosanase was purified, and its molecular weight was estimated to be 31 kDa by SDS-PAGE. Csn-PD displayed maximal activity toward colloidal chitosan at pH 7.0 and 45 °C, respectively. A combination of thin-layer chromatography and electrospray ionization-mass spectrometry results showed that Csn-PD exhibited an endotype cleavage pattern and hydrolyzed chitosan to yield (GlcN)2 as the major product. The unique enzymatic properties of this chitosanase may make it a good candidate for (GlcN)2 production.