Online coupling of thin layer chromatography with matrix‐assisted laser desorption/ionization time‐of‐flight mass spectrometry: synthesis and application of a new material for the identification of carbohydrates by thin layer chromatography/matrix free material enhanced laser desorption/ionization mass spectrometry

This article describes the online hyphenation of thin layer chromatography with matrix free material enhanced laser desorption/ionization mass spectrometry (mf‐MELDI‐MS), the preparation of new material for MELDI and application of this newly synthesized material using TLC/MELDI‐MS for the analysis of carbohydrate reference standards and plant extracts. Samples included within these analyses are standard solutions of glucose, sucrose, raffinose and a plant extract of Quercus robur, which is used for its anti‐inflammatory, anti‐viral and anthelminitc properties in phytomedicine. A new material for mf‐MELDI‐MS is prepared by immobilizing bradykinin – a peptide, on silica gel coupled to 4‐(3‐triethoxysilylpropylureido)azobenzene. This modification enables the absorption of laser energy sufficient for desorption and ionization of low molecular weight molecules like carbohydrates and amino acids. The newly synthesized material delivered excellent results in respect to signal‐to‐noise (S/N) ratio (S/N ratio: >9/1) and sensitivity (limit of detection (LOD): lower to ng/µL). Hyphenation of TLC to MELDI‐MS employing the novel developed material simultaneously as chromatographic and mass spectrometric sorbent was shown for the first time for the analysis of low molecular weight molecules like mono‐ and oligosaccharides. Copyright © 2010 John Wiley & Sons, Ltd.     

Differently complex oligosaccharides can be easily identified by matrix-assisted laser desorption and ionization time-of-flight mass spectrometry directly from a standard thin-layer chromatography plate

Thin-layer chromatography (TLC) is a simple, fast and inexpensive separation method. Unambiguous identification of the TLC spots is, however, often a problem. Here we show for the first time that oligosaccharides (derived from dextran, alginate, hyaluronan and chondroitin sulfate) can be characterized by matrix-assisted laser desorption and ionization time-of-flight (MALDI-TOF) mass spectrometry (MS) directly on a TLC plate. The applied oligosaccharides were either commercially available or obtained from the polysaccharides by HCl-induced hydrolysis. Normal phase TLC was followed by MALDI-TOF MS subsequent to matrix deposition. It will be shown that high quality mass spectra can be obtained that enable unequivocal assignments. It will also be shown that the high content of formic acid in the solvent system does not confer major problems but is responsible for the partial formylation of the analyte and minor N-acetyl loss from hyaluronan and chondroitin sulfate.     

Matrix‐free thin‐layer chromatography/laser desorption ionization mass spectrometry for facile separation and identification of medicinal alkaloids

Quaternary protoberberine alkaloids belong to a pharmaceutically important class of isoquinoline alkaloids associated with bactericidal, fungicidal, insecticidal and antiviral activities. As traditional medicine gains wider acceptance, quick and robust analytical methods for the screening and analysis of plants containing these compounds attract considerable interest. Thin‐layer chromatography (TLC) combined with matrix‐assisted laser desorption/ionization mass spectrometry (MALDI‐MS) is a powerful technique but suffers from dilution of the TLC bands resulting in decreased sensitivity and masking of signals in the low‐mass region both due to addition of matrix. This study integrates for the first time conventional silica gel TLC and laser desorption ionization mass spectrometry (LDI‐MS) thus eliminating the need for any external matrix. Successful separation of berberine (R_f = 0.56) and palmatine (R_f = 0.46) from Berberis barandana including their identification by MS are demonstrated. Furthermore, a robust electrospray ionization (ESI)‐MS method utilizing residual sample from TLC for quantification of berberine applying selected reaction monitoring and standard addition method is presented. The amount of berberine in the plant root prepared for the study was determined to be 0.70% (w/w). Copyright © 2009 John Wiley & Sons, Ltd.     

Application of Thin-Layer Chromatography to High-Performance Liquid Chromatographic Separation of Steroidal Hormones and Cephalosporin Antibiotics

Abstract

High-performance liquid chromatographic (HPLC) separation of steroidal hormones and cephalosporin antibiotics was investigated by adsorption chromatography and reversed-phase chromatography, respectively.

Prior to the HPLC separation of these pharmaceuticals, silica gel thin-layer adsorption chromatography of steroidal hormones and reversed-phase thin-layer partition chromatography of cephalosporin antibiotics with chemically bonded dimethylsilyl silica gel were performed in order to obtain suitable HPLC separation systems.

In the separation of steroidal hormones, the same binary mobile phase ratios of TLC did not give satisfactory results in HPLC. For the sharp separation in HPLC, solvent strength in the binary solvent mixture used for TLC had to be decreased.

The difference in solvent strength for efficient separation between TLC and HPLC might be attributed to the fact that in HPLC the solvent elution power acts in an isocratic manner while in TLC it acts in a gradient manner.

On the other hand, a correlation of mobility between TLC and HPLC separation for cephalosporin antibiotics was obtained, and the possibility of direct transfer of chromatographic systems from TLC to HPLC for separation of these antibiotics was confirmed.     

Separation and identification of some common isomeric plant triterpenoids by thin-layer chromatography and high-performance liquid chromatography

Chromatographic separation of 10 triterpenoids (α-amyrin, β-amyrin, δ-amyrin, lupeol, lupenon, lupeol acetate, cycloartenol, cycloartenol acetate, ursolic acid, oleanolic acid) and 2 sterols (stigmasterol and β-sitosterol) was studied. The chromatographic techniques included silica gel and reversed-phase (C₁₈ RP) thin-layer chromatography (TLC) and C₁₈ RP high-performance liquid chromatography (HPLC) using UV and mass spectrometric (MS) detection with atmospheric pressure chemical ionization (APCI). The TLC separation of the isomeric triterpenols lupeol, α-amyrin, β-amyrin and cycloartenol was achieved for the first time using C₁₈ RP-HPTLC plates. Cycloartenol could be separated from related compounds only on C₁₈ RP-TLC but not on the C₁₈ RP-HPLC. δ-Amyrin isolated from the tomato fruit surface extract could be separated from other amyrins only by HPLC. Tandem mass spectrometry allowed discrimination between the isomers lupeol, α-amyrin, β-amyrin, δ-amyrin, cycloartenol and between lupeol acetate and cycloartenol acetate. The combination of 3 TLC methods and 2 HPLC methods enables qualitative determination of all 12 compounds and proves to be useful for the analysis of plant extracts. It is recommended that TLC screening on silica gel and C₁₈ RP be performed before HPLC analysis.     

Application of thin‐layer chromatography/infrared matrix‐assisted laser desorption/ionization orthogonal time‐of‐flight mass spectrometry to structural analysis of bacteria‐binding glycosphingolipids selected by affinity detection

Glycosphingolipids (GSLs) play key roles in the manifestation of infectious diseases as attachment sites for pathogens. The thin‐layer chromatography (TLC) overlay assay represents one of the most powerful approaches for the detection of GSL receptors of microorganisms. Here we report on the direct structural characterization of microbial GSL receptors by employment of the TLC overlay assay combined with infrared matrix‐assisted laser desorption/ionization orthogonal time‐of‐flight mass spectrometry (IR‐MALDI‐o‐TOF‐MS). The procedure includes TLC separation of GSL mixtures, overlay of the chromatogram with GSL‐specific bacteria, detection of bound microbes with primary antibodies against bacterial surface proteins and appropriate alkaline phosphatase labeled secondary antibodies, and in situ MS analysis of bacteria‐specific GSL receptors. The combined method works on microgram scale of GSL mixtures and is advantageous in that it omits laborious and time‐consuming GSL extraction from the silica gel layer. This technique was successfully applied to the compositional analysis of globo‐series neutral GSLs recognized by P‐fimbriated Escherichia coli bacteria, which were used as model microorganisms for infection of the human urinary tract. Thus, direct TLC/IR‐MALDI‐o‐TOF‐MS adds a novel facet to this fast and sensitive method offering a wide range of applications for the investigation of carbohydrate‐specific pathogens involved in human infectious diseases. Copyright © 2010 John Wiley & Sons, Ltd.     

Ultrathin-Layer Chromatography (UTLC)

Ultrathin-layer chromatography (UTLC) differs from high-performance thin-layer chromatography (HPTLC) and from thin-layer chromatography (TLC) in two basis things: the layer thickness, and the migration distances of the analytes. UTLC has a monolithic or a nanostructured stationary silica gel phase bound directly to the glass plates. Layer thickness in UTLC is 10 μm, instead of 100–250 μm in HPTLC. Migration distances are in the range of 1–3 cm for UTLC, instead of 8–10 cm for HPTLC. Therefore, the major advantages of UTLC over HPTLC and TLC are the shorter development times and higher separation efficiency and sensitivity. Moreover, separations on UTLC plates require smaller reagent and sample volumes. However, the UTLC plates are very difficult to manage with the TLC and HPTLC equipment currently available. Therefore, the next challenge in this area is the development of an inexpensive solution with appropriate instrumentation (sensitive optical scanners and sample application systems). UTLC had been used for separations of many compounds, e.g., pharmaceutically active ingredients, pesticides, plasticisers, natural products, and other chemical substances.     

HPTLC Determination of Cefpodoxime Proxetil in Formulations

An HPTLC method has been developed that coelutes both the isomers of cefpodoxime proxetil (CFP). CFP was chromatographed on a silica gel 60 F₂₅₄ TLC plate using toluene:acetonitrile (6:4) as a mobile phase and was quantified at 234 nm. The method was validated with respect to linearity, accuracy, precision and specificity. The limit of detection and limit of quantification for CFP were found to be 0.150 and 0.4 μg spot⁻¹, respectively. The proposed method was successfully used to determine the amount of CFP present in the marketed tablets and self-nanoemulsifying systems.

     

Normal Phase TLC Separation of Enantiomers Using Chiral Ion Interaction Agents

Abstract

A method for the thin layer chromatographic (TLC) separation of enantiomers and diastereomers involving the use of chiral ion interaction agents is described. Several aromatic amino alcohols were resolved by TLC on diol and/or high performance silica gel plates using a mobile phase containing (1R)-(-)- ammonium-10-camphorsulfonate or N-benzoxycarbonyl-glycyl-L-proline (ZGP). Many of these chiral aromatic amino alcohols are of pharmacological importance as α- and β-adrenergic blockers, adrenergic compounds, and anti-glaucoma agents. A comparison was made between various N-CBZ-amino acid derivatives as chiral counter ions/chiral mobile phase additives (CMAs). These separations could not be achieved on other normal phase TLC stationary phases including microcrystalline cellulose, alumina and ordinary silica gel plates.     

Comparative TOF-SIMS and MALDI TOF-MS analysis on different chromatographic planar substrates

A comparison is made between two high resolution, surface-based, mass spectrometric methods: time-of-flight secondary ion mass spectrometry (TOF-SIMS) and matrix-assisted laser desorption/ionisation mass spectrometry (MALDI TOF-MS) in indication of abietic and gibberellic acids molecular profiles on different chromatographic thin layers. The analytes were applied to silica gel chromatographic thin layers with SIMS on-line interfacing channel, monolithic silica gel ultra-thin layers, and thin layers specifically designed for direct Raman spectroscopic analysis. Two MALDI matrices were used in this research: ferulic acid and 2,5-dihydroxybenzoic acid. The silica gel SIMS-interfacing channel strongly supported formation of numerous different MALDI MS fragments with abietic and gibberellic acids, and ferulic acid matrix. The most intense fragments belonged to [M-OH](+) and [M](+) ions from ferulic acid. Intense conjugates were detected with gibberellic acid. The MALDI MS spectrum from the monolithic silica gel surface showed very low analyte signal intensity and it was not possible to obtain MALDI spectra from a Raman spectroscopy treated chromatographic layer. The MALDI TOF MS gibberellic acid fragmentation profile was shielded by the matrix used and was accompanied by poor analyte identification. The most useful TOF-SIMS analytical signal response was obtained from analytes separated on monolithic silica gel and a SIMS-interfacing modified silica gel surface. New horizons with nanostructured surfaces call for high resolution MS methods (which cannot readily be miniaturised like many optical and electrochemical methods) to be integrated in chip and nanoscale detection systems.     

Analysis of native milk oligosaccharides directly from thin-layer chromatography plates by matrix-assisted laser desorption/ionization orthogonal-time-of-flight mass spectrometry with a glycerol matrix

We have recently presented a new method for direct coupling of high-performance thin-layer chromatography (HPTLC) with matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS), illustrated by the analysis of a complex ganglioside mixture. In the current communication, an adaptation of this procedure to mixtures of native oligosaccharides from human and from elephant milk is described. The key features in this method are (1) glycerol as a liquid matrix, to provide a homogeneous wetting of the silica gel and a simple and fast MALDI preparation protocol, (2) an infrared (IR) laser for volume material ablation and particular soft desorption/ionization conditions, and (3) an orthogonal time-of-flight mass spectrometer for a high mass accuracy, independent of any irregularity of the silica gel surface. Chromatographic "mobility profiles" were determined by scanning the laser beam across the analyte bands. The current limit of detection for the MS analysis was determined to approximately 10 pmol of individual oligosaccharides spotted for chromatography. A liquid composite matrix, containing glycerol and the ultraviolet (UV-)MALDI matrix alpha-cyano-4-hydroxycinnamic acid, allows a direct HPTLC-MALDI-MS analysis with a 337 nm-UV laser as well. Compared to the IR-MALDI mode, the analytical sensitivity in UV-MALDI was found to be lower by one order of magnitude, whereas unspecific analyte ion fragmentation as well as adduct formation was found to be more extensive.     

Densitometric high-performance thin-layer chromatography methods for the quantification of oleuropein in Olea europaea leaves and pharmaceutical preparation utilizing normal- and reversed-phase silica gel plates

Two validated, simple and precise densitometric high-performance thin-layer chromatography (HPTLC) quantification methods were proposed for both qualitative and quantitative estimation of oleuropein in Olea europaea leaves and a pharmaceutical product utilizing normal-phase and reversed-phase silica gel TLC plates. In method I, 10 × 20 cm glass plates coated with 0.2 mm thin layers of normal-phase silica gel 60 containing F₂₅₄ (E-Merck, Germany) and a mixture of ethyl acetate‒methanol‒water (8:1:0.5, V/V) were used as the stationary and the mobile phase, respectively. Method II utilized 10 × 20 cm glass-backed plates supporting 0.2 mm layers of RP-18 silica gel 60 containing F₂₅₄ (E-Merck, Germany) as the stationary phase and green solvents mixture composed of ethanol‒water (5.5:4.5, V/V) as the mobile phase. The two methods resulted in sharp, symmetrical, well-resolved peaks at R_F values of 0.47 ± 0.02 and 0.78 ± 0.03 with linearity ranges 200‒1400 ng/spot (r² = 0.9994) and 200‒1400 ng/spot (r² = 0.9996) for method I and method II, respectively. Spots corresponding to oleuropein were scanned at 200 nm. The two methods complied with the ICH guidelines for validation. Due to simplicity, low cost and short analysis time, the methods can be good alternatives for the quality control of different products containing olive leaves extract or pure oleuropein.

     

Sequencing of oligosaccharides derivatized with benzylamine using electrospray ionization-quadrupole time of flight-tandem mass spectrometry

Oligosaccharides were derivatized by reductive amination using benzylamine and analyzed by nanoelectrospray ionization-quadrupole time of flight-tandem mass spectrometry (nanoESI-QTOF-MS/MS) in the positive ion mode. The major signals were obtained under these conditions from the [M+H]+ ions for all benzylamine-derivatized oligosaccharides. To obtain structural information from these derivatized oligosaccharides, MS/MS was applied. Protonated molecular ions underwent extensive fragmentation, even under low-energy collision-induced dissociation. MS/MS spectra of [M+H]+ ions are characterized by simple fragmentation patterns which result from cleavage of the glycosidic bonds and thus allow a straightforward interpretation. Fragmentation of the [M+H]+ ions gave predominantly B- and Y-type glycosidic fragments. A systematic study of various oligosaccharides showed that information on sugar sequence and branching could easily be obtained. Predictable and reproducible fragmentation patterns could be obtained in all cases. This derivatization procedure and mass spectrometric methodology were applied successfully to neutral and acidic glycans released from 10 microg of glycoproteins separated by gel electrophoresis. Moreover, the derivatives retain their sensitivity to exoglycosidases. Thus a series of sequential on-target exoglycosidase treatments combined with matrix-assisted laser desorption/ionization-time of flight-mass spectrometry (MALDI-TOF-MS) was found to be useful for the determination of structural features of the glycans released from proteins separated by gel electrophoresis such as the monosaccharide sequence, branching pattern, and anomeric configurations of the corresponding glycosidic linkages. Our strategy can be used successfully to assign the major glycans released from proteins separated by gel electrophoresis.     

Transfer of silica gel TLC screening methods for clarithromycin,azithromycin, and amodiaquine + artesunate to HPTLC–densitometry with detection by reagentless thermochemical activation of fluorescence quenching

ABSTRACT

A previously published model process was used for the transfer of silica gel thin-layer chromatography (TLC) screening methods for clarithromycin, azithromycin, and amodiaquine?+?artesunate pharmaceutical formulations published in the Global Pharma Health Fund E.V. Minilab manual for the identification of counterfeit drugs to high-performance TLC–densitometry quantitative methods that can be used in support of regulatory compliance actions. In these new methods, detection of clarithromycin, azithromycin, and artesunate was achieved by thermochemical activation involving simple reagent-free heating of the layer to produce derivatives of the drugs that quench fluorescence under 254?nm ultraviolet light. Additional drugs with TLC screening methods published in the Minilab manual and/or Compendium of Unofficial Methods for Rapid Screening of Pharmaceuticals by Thin Layer Chromatography that do not naturally quench fluorescence were studied and also found to be detectable by thermochemical activation on silica gel layers. The conditions of the thermochemical activation were studied, and in situ spectra of drug zones before and after heating were obtained. Heat activation of fluorescence quenching seems to be a widely applicable detection method that is safer and more convenient than the use of chemical spray, dip, or vapor phase reagents.     

辽东楤木叶中皂苷Congmuyenoside B(2)的分离与鉴定

本研究实现了对辽东楤木叶中Congmuyenoside B(2)的分离与鉴定。研究中采用大孔吸附树脂提取法和硅胶柱层析法对其分离,应用HPLC色谱法和薄层色谱法对纯度进行分析,利用电喷雾多级串联质谱结合核磁共振和红外光谱对该化合物的结构进行分析鉴定。本提取分离法操作简便,提高了分离化合物的纯度。HPLC色谱法灵敏度高,结果准确、减少了质谱及其它色谱中的杂质峰对化合物鉴定的干扰,结构准确,可靠。     

Thin Layer Chromatographic Screening of Coal Liquids

Abstract

Procedures are described for the thin layer chromatographic (TLC) analysis of coal-derived liquids. After solvent selection using Selectosol (1) radial TLC, a series of compounds typically found in coal liquids was used to demonstrate their elution behavior on silica gel. It was determined that unactivated (Brockman 4) silica plates series developed with isooctane/tetrahydrofuran (THF) (80:20, v/v) provided the best separation. The order of elution was saturate >hydroaromatic >polynuclear aromatic (PNA)>phenols ≥ nitrogen bases. The use of two-dimensional TLC with reverse phase and silica contiguous on the same plate (Whatman Multi-K) is discussed for the rapid separation and identification of coal liquid components. Trimethylsilyl ether derivatives were prepared of phenols and alcohols present in coal conversion products. This technique proved most useful in that the TMS-derivatives behaved similarly to their parent PNA, in order of elution, on silica plates using a moderately active mobile phase (isooctane: THF,80:20, v/v). TLC and the reactions described provide a semi-quantitative measure of the degree of hydrogenation of coal-derived liquids.     

TLC Based Method for Standardization of Pongamia pinnata (Karanj) Using Karanjin as Marker

Pongamia pinnata Linn. (Papilionaceae) seeds have gained great commercial and industrial importance owing to their high oil content. Presently, there is no appropriate TLC based method available for standardization of P. pinnata. A simple, robust and reproducible TLC method for the determination of Karanjin is reported in the seeds of P. pinnata. The method involves separation of components by TLC on pre-coated silica gel G 60 F₂₅₄ plates developed on toluene: ethyl acetate (7:3 v/v) and detection at 260 nm in absorbance mode. The sensitivity of the method was found to be 100 ng. The linearity range was 50–300 ng. Four samples of P. pinnata from different geographical locations were tested for their karanjin content using the developed method. The proposed method was found to be robust, precise, and accurate, it therefore holds potential for detection, monitoring and quantification of karanjin in Pongamia pinnata.

     

Thin layer chromatography–spray mass spectrometry: a method for easy identification of synthesis products and UV filters from TLC aluminum foils

A straightforward procedure for direct mass spectrometric (MS) analysis of spots from thin layer chromatography (TLC) plates, without the need of an external ion source, was developed using the aluminum plate backing as spray tip. The spots were cut out shaped as a tip with a 60° angle, mounted in front of the MS orifice, and after addition of a spray solvent spectra were obtained immediately. A high-resolution time-of-flight MS was used since the method is of particular interest for rapid identification or confirmation of spots from TLC plates. The practical benefits of this technique were demonstrated by detection of by-products of organic reactions, by identification of degradation products, and by accurate confirmation of spots when UV filters in sunscreens were analyzed by TLC. Employing the described method TLC spots can be evaluated fast without the need of an external ion source or devices for analyte transfer from TLC to MS, only a basic MS instrument and a high-voltage power supply is required.

Hydroxylation of the silica in microfabricated thin layer chromatography plates as probed by time‐of‐flight secondary ion mass spectrometry and diffuse reflectance infrared Fourier transform spectroscopy

Microfabricated silica thin layer chromatography (TLC) plates have previously been prepared on patterned carbon nanotube forests. The high temperatures used in their fabrication reduce the number of hydroxyl groups on their surfaces. Fortunately, silica can be rehydroxylated. In diffuse reflectance infrared Fourier transform spectroscopy (DRIFT), a silanol peak below 3740 cm^?1 indicates a well‐hydroxylated silica surface that is fit for chromatography. Hydroxylations of our materials with HF are so effective that it is not possible to discern the position of this peak. In contrast, this signal is discernable when the plates are treated with NH₄OH. To find a more convenient method for studying the surfaces of TLC plates, time‐of‐flight secondary ion mass spectroscopy (ToF‐SIMS) was considered. ToF‐SIMS is advantageous because multiple microfabricated TLC plates must be scraped to obtain enough silica for one DRIFT analysis, while static SIMS can be performed on very small regions (500 × 500 µm² or less) of individual plates. Ratios of the SiOH⁺ and Si⁺ ToF‐SIMS signals for microfabricated TLC plates correlated well with ~3740 cm^?1 silanol peaks from DRIFT. Thus, SIMS allows direct analysis of all of our treated and untreated plates, including those hydroxylated with HF. The best hydroxylation condition for HF, which was better than any studied for NH₄OH, was around 150 ppm at room temperature. The best hydroxylation condition for NH₄OH was 50 °C for 72 h. ToF‐SIMS versus DRIFT results of commercial TLC plates were also obtained and evaluated. Copyright © 2014 John Wiley & Sons, Ltd.     

N-linked oligosaccharide analysis of rat brain Thy-1 by liquid chromatography with graphitized carbon column/ion trap-Fourier transform ion cyclotron resonance mass spectrometry in positive and negative ion modes

We have previously described the site-specific glycosylation analysis of rat brain Thy-1 by LC/multistage tandem mass spectrometry (MS(n)) using proteinase-digested Thy-1. In the present study, detailed structures of oligosaccharides released from Thy-1 were elucidated by mass spectrometric oligosaccharide profiling using LC/MS with a graphitized carbon column (GCC-LC/MS). First, using model oligosaccharides, we improved the oligosaccharide profiling by ion trap mass spectrometry (IT-MS) coupled with Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR-MS). Sequential scanning of a full MS(1) scan with FT-ICR-MS followed by data-dependent MS(n) with IT-MS in positive ion mode, and a subsequent full MS(1) scan with FT-ICR-MS followed by data-dependent MS(n) with IT-MS in negative ion mode enabled the monosaccharide composition analysis as well as profiling and sequencing of both neutral and acidic oligosaccharides in a single analysis. The improved oligosaccharide profiling was applied to elucidation of N-linked oligosaccharides from Thy-1 isolated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. It was demonstrated that Thy-1 possesses a significant variety of N-linked oligosaccharides, including Lewis a/x, Lewis b/y, and disialylated structure as a partial structure. Our method could be applicable to analysis of a small abundance of glycoproteins, and could become a powerful tool for glycoproteomics.