A novel approach of combining thin-layer chromatography with surface-assisted laser desorption/ionization (SALDI) time-of-flight mass spectrometry

A novel means of combining thin-layer chromatography (TLC) with laser desorption/ionization mass spectrometry using a liquid matrix is proposed. Surface-assisted laser desorption/ionization (SALDI) mass spectrometry, which uses a mixture of a micrometer-sized carbon powder (graphite or activated carbon, the SALDI solid) and 15% sucrose/glycerol, dissolved in an equal volume of methanol (SALDI liquid) as a SALDI matrix, is used for laser desorption mass analysis. The ablation of carbon powder from a pencil drawing was used as an alternative to the SALDI solid. The liquid matrix resembled that used in a conventional SALDI matrix system. A line was drawn before separation with a pencil on the track of the sample developed on the TLC plate. After TLC separation, approximately 0.1 microl of SALDI liquid was directly applied to the chromatographic spots on the TLC plate. Porphyrins were used to demonstrate this combination owing to the visible colors of this type of compound. The analyte signal can be easily detected by irradiating the laser along the pencil line on the TLC plate. An additive, p-toluenesulfonic acid, is added to the SALDI liquid to enhance the signal's intensity. This additive dramatically improves the signal-to-noise ratio. A detection limit of approximately 500 pg is demonstrated for porphines, which is 50 times better than that corresponding to conventional TLC SALDI.     

薄层色谱与质谱联用的研究进展

薄层色谱(TLC)是一类非常实用的液相色谱方法,由于其装置简单、操作便捷、灵活、通量高、成本低,以及样品前处理简单等优点,在许多行业的检测中都有广泛的应用并扮演着重要的角色。随着现代检测技术的不断发展以及各种检测技术综合应用程度的加深,薄层色谱与质谱的联用(TLC-MS)也成为这一方法的重要发展方向。随着我国医药、食品、科学仪器等事业的不断发展和升级,相信薄层色谱-质谱联用技术可以起到更好的作用,并迎来发展的契机。该综述将目前薄层色谱-质谱的联用形式分为3类,一是接口仪器的间接联用,二是质谱对薄层板的原位检测,三是质谱对薄层分离过程的实时监测,并按此分类对典型的联用形式进行了总结和简要描述。随着薄层色谱-生物自显影技术的广泛使用,薄层色谱与质谱联用的技术方法极大地提高了食品、药用生物活性物质的研发效率。目前,薄层色谱与质谱联用发展的主要瓶颈是“即插即用”型部件的设计和商品化。具有实时监测功能,同时又兼备灵活扫描功能和高通量特点的TLC-MS技术也很令人期待。此外,不同种类TLC-MS解吸-电离技术的对比研究也是有待讨论的应用问题。     

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.     

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.     

Analysis of Glycosaminoglycan Oligosaccharides by Combined HPTLC/MALDI-TOF MS: Reduced Silica Gel Thickness Leads to Improved Spectral Qualities and Reduced Side Reactions

Thin-layer chromatography (TLC) is a simple, fast and inexpensive separation method which can be applied to virtually all natural products including oligosaccharides. Unfortunately, however, the unequivocal identification of a TLC spot is normally difficult. Fortunately, this problem can be minimized when mass spectrometry (MS) such as matrix-assisted laser desorption and ionization time-of-flight is used to identify the TLC spots. This work is dedicated to the TLC/MS analysis of oligosaccharides derived from native chondroitin sulfate and hyaluronan. We will show that the thickness of the silica gel layer (200 versus 100 µm) has a tremendous influence on the quality of the mass spectra: a reduced silica gel thickness enhances the spectral quality and, in particular, improves the achievable signal-to-noise ratio. Additionally, unwanted formylation of the GAG oligosaccharides (which occurs due to the high moiety of formic acid in the mobile phase) can also be minimized if MS-grade HPTLC plates are used.

     

Ganglioside analysis by thin-layer chromatography matrix-assisted laser desorption/ionization orthogonal time-of-flight mass spectrometry

Metastable decomposition of ions generated in matrix-assisted laser desorption/ionization (MALDI) mass spectrometers complicates analysis of biological samples that have labile bonds. Recently, several academic laboratories and manufacturers of commercial instruments have designed instruments that introduce a cooling gas into the ion source during the MALDI event and have shown that the resulting vibrational cooling stabilizes these labile bonds. In this study, we compared stabilization and detection of desorbed gangliosides on a commercial orthogonal time-of-flight (oTOF) instrument with results we reported previously that had been obtained on a home-built Fourier transform mass spectrometer. Decoupling of the desorption/ionization from the detection steps resulted in an opportunity for desorbing thin-layer chromatography (TLC)-separated gangliosides directly from a TLC plate without compromising mass spectral accuracy and resolution of the ganglioside analysis, thus coupling TLC and oTOF mass spectrometry. The application of a declustering potential allowed control of the matrix cluster and matrix adduct formation, and, thus, enhanced the detection of the gangliosides.     

MALDI-TOF-MS Directly Combined with TLC: A Review of the Current State

Thin-layer chromatography (TLC) is a widely used, fast and inexpensive method for separating complex mixtures. Unfortunately, the quality of achievable separation represents only one side. An additional problem is the unambiguous assignment of the obtained spots to defined compounds. Clear identification of spots is often not possible by common staining methods and comparison with a known reference compound. Therefore, further analytical techniques are mostly required for further structural elucidation. Mass spectrometry (MS) is a suitable method due to its high sensitivity. In particular, matrix-assisted laser desorption and ionization time-of-flight (MALDI-TOF) MS is a modern soft-ionization technique that may be easily combined with TLC. This review summarizes the so far available knowledge about direct TLC–MALDI combination and gives an overview about different molecule classes that have already been successfully analyzed by this approach. This review critically summarizes the capabilities and limitations of the direct MALDI–TLC combination and highlights in particular the problems related to sample preparation and instrumentation.     

Characterization of polyether mixtures using thin-layer chromatography and matrix-assisted laser desorption/ionization mass spectrometry

Thin-layer chromatography (TLC) and matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) were combined to achieve characterization of polyether mixtures. Three polyethers, polyethylene glycol (PEG), polypropylene glycol (PPG) and polytetramethylene glycol (PTMG), or mixtures of these compounds, were studied. One shortcoming of mixture analysis of synthetic polymers using MALDI-MS is that individual polymers in the mixture may display different detection sensitivities. For example, the MALDI mass spectrum of an equimolar mixture of PEG, PPG and PTMG displayed a high intensity of PPG ions, while no PTMG ions were detectable; however, PTMG ions were detected after the mixture had been separated by TLC. This combined TLC and MALDI-MS analysis of a PPG polymer bearing reactive epoxy groups showed that the polymer contained byproducts with different end-groups. These byproducts were identified as chloro-substituted polymers formed during polymer synthesis. Our study shows TLC to be a rapid and low-cost separation technique, and that it can be combined with MALDI-MS to achieve effective analysis of synthetic polymers.     

MALDI-TOF-MS Directly Combined with TLC: A Review of the Current State

Thin-layer chromatography (TLC) is a widely used, fast and inexpensive method for separating complex mixtures. Unfortunately, the quality of achievable separation represents only one side. An additional problem is the unambiguous assignment of the obtained spots to defined compounds. Clear identification of spots is often not possible by common staining methods and comparison with a known reference compound. Therefore, further analytical techniques are mostly required for further structural elucidation. Mass spectrometry (MS) is a suitable method due to its high sensitivity. In particular, matrix-assisted laser desorption and ionization time-of-flight (MALDI-TOF) MS is a modern soft-ionization technique that may be easily combined with TLC. This review summarizes the so far available knowledge about direct TLC–MALDI combination and gives an overview about different molecule classes that have already been successfully analyzed by this approach. This review critically summarizes the capabilities and limitations of the direct MALDI–TLC combination and highlights in particular the problems related to sample preparation and instrumentation.

     

Analysis of Planar Chromatograms by Fast Atom Bombardment and Laser Desorption Mass Spectrometry

Abstract

The applications of fast atom bombardment mass spectrometry coupled with thin-layer chromatography in a study of phospholipids, alkaloids derived from bloodroot rhizomes, and diuretics are summarized. Use of the mass spectrometric data to trace compounds via characteristic fragment ions increases the effective resolution of the chromatographic resolution. Quantitative values derived in TLC/FAB experiments for the diuretic amiloride hydrochloride is linear over two orders of magnitude encompassing the clinically useful range. The use of a laser desorption ionization method and Fourier transform mass analysis for TLC plate detection is also described; matrix and concentration effects are significant in this case, but several successful applications are described. An interface device for the linkage     

Interfacing matrix-assisted laser desorption/ionization mass spectrometry with column and planar separations

This paper presents a critical review of off-line and on-line coupling of matrix-assisted laser desorption/ionization (MALDI) mass spectrometry to liquid column separations (e.g. HPLC, GPC and CE) and planar separations (e.g. PAGE and TLC). Off-line MALDI analysis of fractions collected from HPLC, GPC and CE or spots scraped and extracted from TLC and PAGE has already become a routine practice for many laboratories. MALDI has also been used to obtain mass spectra directly from TLC plates and PAGE. The direct analysis methods range from dot-blotted samples to two-dimensional scanning of the entire gels/plates. Various combinations of on-line coupling of MALDI with column separations are also reviewed. The review discusses the strengths and limitations associated with different off-line and on-line coupling approaches.     

Interfacing matrix-assisted laser desorption/ionization mass spectrometry with column and planar separations

This paper presents a critical review of off-line and on-line coupling of matrix-assisted laser desorption/ ionization (MALDI) mass spectrometry to liquid column separations (e.g. HPLC, GPC and CE) and planar separations (e.g. PAGE and TLC). Off-line MALDI analysis of fractions collected from HPLC, GPC and CE or spots scraped and extracted from TLC and PAGE has already become a routine practice for many laboratories. MALDI has also been used to obtain mass spectra directly from TLC plates and PAGE. The direct analysis methods range from dot-blotted samples to two-dimensional scanning of the entire gels/plates. Various combinations of on-line coupling of MALDI with column separations are also reviewed. The review discusses the strengths and limitations associated with different off-line and on-line coupling approaches.     

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.     

Mass spectrometric analysis of ceramide composition in mono-, di-, tri-, and tetraglycosylceramides from mouse kidney: an experimental model for uropathogenic Escherichia coli.

Many bacteria have been shown to bind to the carbohydrate part of glycosphingolipids, but also the lipid moieties of receptor-active glycolipids are of importance. To investigate the chemistry of the ceramides of kidney glycolipids to which the uropathogenic Escherichia coli bind, different mass spectrometric techniques were utilized. First, a mixture of glycolipids isolated from man and mice kidney was separated by thin-layer chromatography (TLC) and scanned by direct desorption from the plate by fast atom bombardment mass spectrometry (TLC/FAB-MS). Second, the glycolipids were purified by preparative TLC and analyzed by negative-ion FAB-MS. After methylation, further analyses were made with positive-ion FAB-MS, positive-ion electron ionization (EI)-MS, high-temperature capillary gas chromatography (GC/EI-MS) and positive-ion matrix-assisted laser desorption/ionization (MALDI)-MS. The ceramide compositions of the four glycolipids were determined using all these MS techniques and the reliability of the different methods for this type of analyses is discussed. Comparison of the mouse kidney glycolipids with the corresponding glycolipids from human kidney showed the same degree of hydroxylation of ceramides among mono- and disaccharide glycolipids, but a significantly higher degree of hydroxylation among mouse kidney glycolipids with three and four sugar residues. This result might be of relevance for the binding of P-fimbriated E. coli to the urinary tract tissues.     

Rapid identification of siderophores by combined thin-layer chromatography/matrix-assisted laser desorption/ionization mass spectrometry

The investigation of a combined thin-layer chromatography/matrix-assisted laser desorption/ionization mass spectrometry (TLC/MALDI-MS) method for the analysis of siderophores from microbial samples is described. The investigated siderophores were enterobactin, ferrioxamine B, ferrichrome, ferrirhodin, rhodotorulic acid and coprogen. Solid-phase extraction was employed to recover the siderophores from the microbial samples. After visualization of the spots via spraying with ferric chloride or chrome azurol sulfonate assay solution, the MALDI matrix was applied to the gel surface. Several TLC/MALDI experimental parameters were optimized, such as type and concentration of MALDI matrix, as well as the type and composition of solvent to facilitate analyte transport from the inside of the TLC gel to the surface. The impact of these parameters on sensitivity, precision and ion formation of the various siderophores was studied. The detection limits for the investigated siderophores were in the range 1-4 pmol. These values were about 4-24 times higher than the detection limits obtained directly from stainless steel MALDI targets. The differences were most likely due to incomplete transport of the 'trapped' analyte molecules from the deeper layers of the TLC gel to the surface and into the matrix layer. In addition, chromatographic band broadening spread the analyte further in TLC as compared with the steel plates, resulting in less analyte per surface area. The identification of the siderophores was aided by concurrently applying a Ga(III) nitrate solution to the TLC plate during the visualization step. The resulting formation of Ga(III) complexes lead to distinctive (69)Ga/(71)Ga isotope patterns in the mass spectra. The versatility of the TLC/MALDI-MS assay was demonstrated by using it to analyze siderophores in a Pseudomonas aeruginosa sample. An iron-binding compound was identified in the sample, namely pyochelin (2-(2-o-hydroxyphenyl-2-thiazolin-4-yl)-3-methylthiazolidine-4-carboxylic acid).     

High pressure matrix-assisted laser desorption/ionization Fourier transform mass spectrometry for minimization of ganglioside fragmentation

Transiently elevating pressure in a matrix-assisted laser desorption/ionization Fourier transform mass spectrometry (MALDI-FTMS) source into the 1-10 mbar range during ionization decreases the metastable fragmentation of gangliosides. This allows detection of the molecular ion species without loss of the highly labile sialic acid residues. In these experiments, gangliosides with up to five sialic acids were ionized by MALDI and detected with the FTMS. In each case, when the high pressure collisional cooling was used, the singly charged molecular ion was the base peak in the spectra, both in the positive and negative ion modes, and minimal metastable fragmentation was observed. This result is promising, as the previously developed TLC separation methods can be coupled to MALDI-FTMS.     

Laser Desorption Ionization of Melamine and Identification of Fragmentation Channels from Ion Velocity Distribution Measurements

Fragmentation frequently accompanies intact laser desorption ionization of a parent non-volatile compound, desorption and dissociation dynamics has been a subject of intense studies over the past decade. As a preliminary lest system for future laser desorption study of energetic compounds such as explosives and propellents, we studied UV laser desorption ionization of melamine at a laser power density of approximately 4.4 MW/cm². Several gas-phase dissociation channels of the parent and fragment ions formed in UV laser desorption ionization of melamine films can be identified from their velocity distributions. A phenomenological desorption temperature of the order of 20000 K is estimated from fitting the experimental velocity distributions to Maxwellian functions.     

Coupling of planar chromatography with Direct Analysis in Real Time mass spectrometry

Direct Analysis in Real Time mass spectrometry (DART-MS) is an emerging and rapidly developing area of ambient desorption ionization mass spectrometric techniques. Its coupling with planar chromatography is especially promising, as compared to other ambient desorption ionization techniques, because it does not require the use of liquids that may distort the shape of a spot by diffusion effects. In the first publications on TLC/HPTLC-DART-MS, due to the fixed, horizontally aligned supply of the gas flow from the DART ionization source to the MS inlet, the introduction of HPTLC/TLC plates as cut strips was inconvenient for quantitation, and the repeatability was very low due to the manual positioning. Recently a new version of the DART ion source was suggested, which allows adjusting the angle of the DART gas stream and the use of a motorized rail, thereby, improving highly the capabilities of TLC/HPTLC-DART-MS. This comprehensive review describes the development and analytical capabilities of TLC/HPTLC-DART-MS, and the general DART-MS perspectives for surface analysis or imaging MS.      

A direct and simple method of coupling matrix-assisted laser desorption and ionization time-of-flight mass spectrometry (MALDI-TOF MS) to thin-layer chromatography (TLC) for the analysis of phospholipids from egg yolk

Although the most important application of matrix-assisted laser desorption and ionization time-of-flight mass spectrometry (MALDI-TOF MS) is "proteomics," there is growing evidence that this soft ionization method is also useful for phospholipid (PL) analysis. Although all PLs are detectable by MALDI-TOF MS, some lipid classes, particularly those with quaternary amines such as phosphatidylcholines (PCs), are more sensitively detected than others, and these suppress the signals of less sensitively detected PLs when complex mixtures are analyzed. Therefore, a separation of the total organic extract into individual lipid classes is necessary. As MALDI uses a solid sample, the direct evaluation of thin-layer chromatography (TLC) plates is possible. We report here on a method of directly coupling MALDI-TOF MS and TLC that can be easily implemented on commercially available MALDI-TOF devices. A total extract of hen egg yolk is used as a simple PL mixture to demonstrate the capabilities of this method. It will be shown that "clean" spectra without any major contributions from fragmentation products and matrix peaks can be obtained, and that this approach is even sensitive enough to detect the presence of PLs at levels of less than 1% of the total extract.     

Rapid,simple, and highly sensitive analysis of drugs in biological samples using thin-layer chromatography coupled with matrix-assisted laser desorption/ionization mass spectrometry

Rapid and precise identification of toxic substances is necessary for urgent diagnosis and treatment of poisoning cases and for establishing the cause of death in postmortem examinations. However, identification of compounds in biological samples using gas chromatography and liquid chromatography coupled with mass spectrometry entails time-consuming and labor-intensive sample preparations. In this study, we examined a simple preparation and highly sensitive analysis of drugs in biological samples such as urine, plasma, and organs using thin-layer chromatography coupled with matrix-assisted laser desorption/ionization mass spectrometry (TLC/MALDI/MS). When the urine containing 3,4-methylenedioxymethamphetamine (MDMA) without sample dilution was spotted on a thin-layer chromatography (TLC) plate and was analyzed by TLC/MALDI/MS, the detection limit of the MDMA spot was 0.05 ng/spot. The value was the same as that in aqueous solution spotted on a stainless steel plate. All the 11 psychotropic compounds tested (MDMA, 4-hydroxy-3-methoxymethamphetamine, 3,4-methylenedioxyamphetamine, methamphetamine, p-hydroxymethamphetamine, amphetamine, ketamine, caffeine, chlorpromazine, triazolam, and morphine) on a TLC plate were detected at levels of 0.05 − 5 ng, and the type (layer thickness and fluorescence) of TLC plate did not affect detection sensitivity. In addition, when rat liver homogenate obtained after MDMA administration (10 mg/kg) was spotted on a TLC plate, MDMA and its main metabolites were identified using TLC/MALDI/MS, and the spots on a TLC plate were visualized by MALDI/imaging MS. The total analytical time from spotting of intact biological samples to the output of analytical results was within 30 min. TLC/MALDI/MS enabled rapid, simple, and highly sensitive analysis of drugs from intact biological samples and crude extracts. Accordingly, this method could be applied to rapid drug screening and precise identification of toxic substances in poisoning cases and postmortem examinations.