Ultra-thin-layer chromatography mass spectrometry and thin-layer chromatography mass spectrometry of single peptides of angiotensin-converting enzyme inhibitors

The separation of structurally related angiotensin-converting enzyme (ACE) inhibitors lisinopril, cilazapril, ramipril and quinapril and their corresponding active diacid forms (prilates) by conventional TLC silica gel 60 plates was contrasted with that afforded by monolithic ultra-thin-layer chromatographic (UTLC) plates. For the use of UTLC plates technical modifications of the commercially available equipments for the sample application, development and detection were made. Plates were developed in modified horizontal developing chamber using ethyl acetate-acetone-acetic acid-water (4:1:0.25:0.5, v/v). Detection of the separated compounds was performed densitometrically in absorption/reflectance mode at 220 nm and after exposure to iodine also by image analysis. The obtained results showed that monolithic layer is more efficient for the separation of structurally similar polar compounds, such as prilates than conventional silica layers. Identification of the compounds was confirmed by ESI-MS after their on-line extraction from the UTLC and TLC plates by means of Camag TLC-MS interface.     

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.     

Preparation of porous n-type silicon sample plates for desorption/ionization on silicon mass spectrometry (DIOS-MS)

This study focuses on porous silicon (pSi) fabrication methods and properties for desorption ionization on silicon mass spectrometry (DIOS-MS). PSi was prepared using electrochemical etching of n-type silicon in HF-ethanol solution. Porous areas were defined by a double-sided illumination arrangement: front-side porous areas were masked by a stencil mask, eliminating the need for standard photolithography, and backside illumination was used for the backside ohmic contact. Backside illumination improved the uniformity of the porosified areas. Porosification conditions, surface derivatizations and storage conditions were explored to optimize pSi area, pore size and pore depth. Chemical derivatization of the pSi surfaces improved the DIOS-MS performance providing better ionization efficiency and signal stability with lower laser energy. Droplet spreading and drying patterns on pSi were also examined. Pore sizes of 50-200 nm were found to be optimal for droplet evaporation and pore filling with the sample liquid, as measured by DIOS efficiency. With DIOS, significantly better detection sensitivity was obtained (e.g. 150 fmol for midazolam) than with desorption ionization from a standard MALDI steel plate without matrix addition (30 pmol for midazolam). Also the noise that disturbs the detection of low-molecular weight compounds at m/z < 500 with MALDI could be clearly reduced with DIOS. Low background MS spectra and good detection sensitivity at the 100-150 fmol level for pharmaceutical compounds were achieved with DIOS-MS.     

Desorption electrospray ionization mass spectrometry for the analysis of pharmaceuticals and metabolites

The performance of desorption electrospray ionization (DESI) in the analysis of a group of pharmaceuticals and their glucuronic acid conjugates is reported. The suitability of different sprayer solvents and different surfaces was examined. In the positive ion mode, water/methanol/trifluoroacetic acid performed best, whereas, in the negative ion mode, water/methanol/ammonium hydroxide was found to be the most suitable spray solvent. Of the surfaces investigated, polymethylmethacrylate (PMMA) was found to give the best performance in terms of sensitivity. Spray solution flow rate and the distance of the sprayer tip from the surface were also found to have significant effects on the signal intensity. Analytes with basic groups efficiently formed the corresponding protonated molecules in the positive ion mode, whereas acidic analytes, such as the glucuronic acid conjugates, formed intense signals due to the deprotonated molecules in the negative ion mode. Ionization of neutral compounds was less efficient and in many cases it was achieved through adduct formation with simple anions or cations.     

Analysis of small molecules by ultra thin-layer chromatography-atmospheric pressure matrix-assisted laser desorption/ionization mass spectrometry

The feasibility of ultra thin-layer chromatography atmospheric pressure matrix-assisted laser desorption ionization mass spectrometry (UTLC-AP-MALDI-MS) has been studied in the analysis of small molecules. Because of a thinner adsorbent layer, the monolithic UTLC plates provide 10-100 times better sensitivity in MALDI analysis than conventional high performance thin-layer chromatography (HPTLC) plates. The limits of detection down to a low picomole range are demonstrated by UTLC-AP-MALDI-MS. Other advantages of UTLC over HPTLC include faster separations and lower solvent consumption. The performances of AP-MALDI-MS and vacuum MALDI-MS have been compared in the analysis of small drug molecules directly from the UTLC plates. The desorption from the irregular surface of UTLC plates with an external AP-MALDI ion source combined with an ion trap instrument provides clearly less variation in measurements of m/z values when compared with a vacuum MALDI-time-of-flight (TOF) instrument. The performance of the UTLC-AP-MALDI-MS method has been applied successfully to the purity analysis of synthesis products produced by solid-phase parallel synthesis method.     

Aligned electrospun nanofibers for ultra-thin layer chromatography

The fabrication and implementation of aligned electrospun polyacrylonitrile (PAN) nanofibers as a stationary phase for ultra-thin layer chromatography (UTLC) is described. The aligned electrospun UTLC plates (AE-UTLC) were characterized to give an optimized electrospun mat consisting of high nanofiber alignment and a mat thickness of ∼25 μm. The AE-UTLC devices were used to separate a mixture of β-blockers and steroidal compounds to illustrate the properties of AE-UTLC. The AE-UTLC plates provided shorter analysis time (∼2–2.5 times faster) with improved reproducibility (as high as 2 times) as well as an improvement in efficiency (up to100 times greater) relative to non-aligned electrospun-UTLC (E-UTLC) devices.     

Trace detection of inorganic oxidants using desorption electrospray ionization (DESI) mass spectrometry

Desorption electrospray ionization (DESI), an established ambient ionization method in mass spectrometry (MS) for the analysis of organic compounds, is applied here to trace detection of inorganic salts, including inorganic oxidants. In-situ surface analysis of targeted compounds, including nitrogen-, halogen- and sulfur-salts, down to sub-nanogram levels, was performed using DESI-MS. Successful experiments were carried out in both the negative and the positive ion modes; simple anions and cations as well as small cluster ions were observed. Various surfaces are examined and surface porosity effects were briefly explored. Absolute detection limits on porous polytetrafluoroethylene (PTFE) of 120 pg (surface concentration 0.07 ng mm⁻²) and 50 pg (surface concentration 0.03 ng mm⁻²), were achieved for sodium chlorate and sodium perchlorate, respectively. The compounds of interest were examined in the presence of a hydrocarbon mixture to assess matrix effects: only a two- or three-fold decrease in the target ion intensity was observed. Commercial fireworks were analyzed to determine perchlorate salts in complex mixtures. This work demonstrates the potential applicability of ambient ionization mass spectrometry to forensic investigations involving improvised explosives.      

Surface effects and electrochemical cell capacitance in desorption electrospray ionization

Time resolved measurements show that during a desorption electrospray ionization (DESI) experiment, the current initially rises sharply, followed by an exponential decrease to a relatively steady current. When the high voltage on the spray emitter is switched off, the current drops to negative values, suggesting that the direction of current flow in the equivalent DESI circuit is reversed. These data demonstrate that the DESI source behaves as a dc capacitor and that the addition of a surface between the sprayer and the counter electrode in DESI introduces a new electrically active element into the system. The charging and discharging behavior was observed using different surfaces and it could be seen both by making current measurements on a plate at the entrance to the mass spectrometer as well as by measuring ion current in the linear ion trap within the vacuum system of the mass spectrometer. The magnitude of the steady state current obtained without analyte present on the surface is different for different surface materials, and different capacitor time constants of the equivalent RC circuits were calculated for different DESI surfaces. The PTFE surface has by far the greatest time constant and is also able to produce the highest DESI currents. Surface properties play a crucial role in charge transfer during DESI in addition to the effects of the chemical properties of the analyte. It is suggested that surface energy (wettability) is an important factor controlling droplet behavior on the surface. The experimental data are correlated with critical surface tension values of different materials. It is proposed, based on the results presented, that super-hydrophobic materials with extremely high contact angles have the potential to be excellent DESI substrates. It is also demonstrated, using the example of the neurotransmitter dopamine, that the surface charge that develops during a DESI-MS experiment can cause electrochemical oxidation of the analyte.     

Fast analysis of high-energy compounds and agricultural chemicals in water with desorption electrospray ionization mass spectrometry

Novel sampling and detection methods using desorption electrospray ionization (DESI) are examined in the detection of explosives (RDX, TNT, HMX, and TNB) and agricultural chemicals (atrazine, alachlor and acetochlor) from aqueous matrices and authentic contaminated groundwater samples. DESI allows analysis of solid and liquid compounds directly from surfaces of interest with little or no sample preparation. Significant savings in analysis time and sample preparation are realized. The methods investigated here include (i) immediate analysis of filter paper wetted with contaminated water samples without further sample preparation, (ii) rapid liquid-liquid extraction (LLE), and (iii) analyte extraction from contaminated groundwater samples on-site using solid-phase extraction (SPE) membranes, followed by direct DESI analysis of the membrane. The wetted filter paper experiment demonstrates the maximum sample throughput for DESI analysis of aqueous matrices but has inadequate sensitivity for some of these analytes. Both the LLE and the SPE methods have adequate sensitivity. The resulting SPE membranes and/or small volume solvent extracts produced in these experiments are readily transported to off-site facilities for direct analysis by DESI. This realizes a significant reduction in the costs of sample shipping compared with those for typical liter-sized samples of groundwater. Total analysis times for these preliminary DESI analyses are comparable with or shorter than those for GC/MS and limits of detection approach environmental action levels for these compounds while maintaining a modest relative standard deviation. Tandem mass spectrometric data is used to provide additional specificity as needed.     

Carbon nanotube and carbon nanorod-filled polyacrylonitrile electrospun stationary phase for ultrathin layer chromatography

The application of carbon nanotube or nanorod/polyacrylonitrile (PAN) composite electrospun nanofibrous stationary phase for ultrathin layer chromatography (UTLC) is described herein. Multi-walled carbon nanotubes (MWCNTs) and edge-plane carbon (EPC) nanorods were prepared and electrospun with the PAN polymer solution to form composite nanofibers for use as a UTLC stationary phase. The analysis of laser dyes demonstrated the feasibility of utilizing carbon nanoparticle-filled electrospun nanofibers as a UTLC stationary phase. The contribution of MWCNT or EPC in changing selectivity of the stationary phase was studied by comparing the chromatographic behavior among MWCNT–PAN plates, EPC–PAN plates and pure PAN plates. Carbon nanoparticles in the stationary phase were able to establish strong π–π interactions with aromatic analytes. The separation of five polycyclic aromatic hydrocarbons (PAHs) demonstrated enhanced chromatographic performance of MWCNT-filled stationary phase by displaying substantially improved resolution and separation efficiency. Band broadening of the spots for MWCNT or EPC-filled UTLC stationary phases was also investigated and compared with that for pure PAN stationary phases. A 50% improvement in band dispersion was noted using the MWCNT based composite nanofibrous UTLC plates.     

自建电喷雾解吸电离源（DESI）在大分子分析中的应用及离子化机理探索

自建了简易的电喷雾解吸电离源(DESI),优化了DESI源喷口的位置和角度,并将其用于常见多肽和蛋白质的分析。多肽和小质量蛋白质(<20 kDa)可以容易地从表面解吸电离,生成清晰的质谱。而牛血清白蛋白(66.4 kDa)不能产生清晰的多电荷分布的质谱,说明当前DESI源的设计可能存在一个电离的分子量上限。通过比较不同的实验条件并对比ESI-MS,发现溶剂分子的挥发过程对电荷分布以及峰宽均有显著影响,可能是由于ESI更软引起。载样表面的性质对DESI-MS的信号强度有较大影响。金表面的自组装单分子膜(SAM)相对于纯金表面有较好的绝缘性,并有助于产生较强信号,说明来自表面的电子转移(电中和)是电喷雾解吸电离过程中的一个重要因素。该文的研究有助于对DESI-MS的实验条件和载样表面的选择,同时增进了对电喷雾解吸电离机理的了解。     

Fabric analysis by ambient mass spectrometry for explosives and drugs

Desorption electrospray ionization (DESI) is applied to the rapid, in-situ, direct qualitative and quantitative analysis of mixtures of explosives and drugs from a variety of fabrics, including cotton, silk, denim, polyester, rayon, spandex, leather and their blends. The compounds analyzed were explosives: trinitrohexahydro-1,3,5-triazine (RDX), octahydro-1,3,5,7-tetranitro-1,3,5,7-tetrazocine (HMX), 2,4,6-trinitrotoluene (TNT), pentaerythritol tetranitrate (PETN) and the drugs of abuse: heroin, cocaine, and methamphetamine. Limits of detection are in the picogram range. DESI analyses were performed without sample preparation and carried out in the presence of common interfering chemical matrices, such as insect repellant, urine, and topical lotions. Spatial and depth profiling was investigated to examine the depth of penetration and lateral resolution. DESI was also used to examine cotton transfer swabs used for travel security sample collection in the screening process. High throughput quantitative analysis of fabric surfaces for targeted analytes is also reported.     

Desorption electrospray ionization (DESI) mass spectrometry and tandem mass spectrometry (MS/MS) of phospholipids and sphingolipids: Ionization, adduct formation, and fragmentation

Desorption electrospray ionization (DESI) mass spectrometry was evaluated for the characterization of glycerophospholipid standards, including glycerophosphocholine (GPCho), glycerophosphoglycerol (GPGro), glycerophosphoethanolamine (GPEtn), glycerophosphoserine (GPSer), glycerophosphoinositol (GPIns), cardiolipin (CL), and sphingolipid standards, including sulfatides (ST) and sphingomyelin (SM). Of specific interest were the effects of surface and solvent composition on signal stability and intensity, along with the ions observed in the full scan mode and the fragmentations seen upon collisional activation for each of the above classes. These experiments were performed without the addition of matrix compounds to the sample and were conducted in the free ambient environment at atmospheric pressure. The compounds GPSer, GPGro, GPIns, ST, and CL were best analyzed in the negative ion mode while PE was ionized efficiently in both positive and negative ion modes. SM and GPCho, which typically generate more abundant ions in the positive ion mode, could be analyzed in the negative ion mode by the addition of anionic reagents such as acetate to the spray solvent. Full scan DESI mass spectra and tandem (MS/MS) spectra for this representative set of physiological phospho/sphingolipids are presented. Similarities with other ionization methods in terms of fragmentation behavior were strong, although ambient ionization of untreated samples is only available with DESI. The effect of surface and solvent properties on signal intensity and stability were determined by depositing standard compounds on several different surfaces and analyzing with various proportions of methanol in the aqueous spray. Analysis was extended to complex mixtures of phospholipids and sphingolipids by examining the total lipid extract of porcine brain and by direct analysis of rat brain cryotome sections. These types of mixture analyses and molecular imaging studies are likely to represent major areas of application of DESI.     

The wettability of polytetrafluoroethylene and polymethyl methacrylate by aqueous solution of two cationic surfactants mixture

Advancing contact angle (theta) measurements were carried out for aqueous solutions of cetyltrimethylammonium bromide (CTAB) and cetylpyridinium bromide (CPyB) mixtures on polytetrafluoroethylene (PTFE) and polymethyl methacrylate (PMMA). The obtained results indicate that the wettability of PTFE and PMMA by aqueous solutions of CTAB and CPyB mixtures depends on the composition and concentration of the mixture; however, synergism in the wettability does not exist. In the range of low concentrations of aqueous solution mixtures there is a linear dependence between the contact angle and composition of the mixtures, but at a concentration close to CMC a deviation from linear dependence is observed. In contrast to Zisman, there is no linear dependence between costheta and the surface tension of aqueous solution of CTAB and CPyB mixtures, but a linear dependence exists between the adhesional and surface tension, and these lines have a slope -1 and -0.34 for PTFE and PMMA, respectively, which suggests that adsorption of CTAB and CPyB mixtures at water-air and PTFE-water is the same, and the orientation of the CTAB and CPyB molecules at both interfaces in the saturated monolayer should also be the same. Adsorption of these mixtures at water-air interface is considerably higher than at PMMA-water interface, and CTAB and CPyB molecules should be parallelly oriented to PMMA surface in the saturated monolayer. Extrapolation of the straight lines to the points corresponding to the surface tension of aqueous solution, which completely spreads over the PTFE and PMMA surface, gives a critical surface tension of wetting equal to 23.44 and 33.13 mN/m, respectively. The value of 23.44 mN/m is higher than that of the surface tension of PTFE, but the value of 33.13 is lower than that of Lifshitz-van der Waals components of PMMA surface tension. On the basis of the critical surface tension, the surface tension of PTFE and PMMA, the Young equation, and thermodynamic analysis of the adhesion work of aqueous solution of surfactant to polymer surface, it was found that for PTFE and PMMA the changes of the contact angle of aqueous solution of two cationic surfactants mixtures on their surfaces as a function of the solution concentration resulted only from the decrease of the polar component of the solution surface tension.     

Critical parameters for the analysis of anionic oligosaccharides by desorption electrospray ionization mass spectrometry

Sulfated oligosaccharides derived from glycosaminoglycans (GAGs) are fragile compounds, highly polar and anionic. We report here on the rare but successful application of desorption electrospray ionization (DESI) — LTQ‐Orbitrap mass spectrometry (MS) to the high‐resolution analysis of anionic and sulfated oligosaccharides derived from the GAGs hyaluronic acid and heparin. For that purpose, key parameters affecting DESI performance, comprising the geometric parameters of the DESI source, the probed surface and the spraying conditions, applied spray voltage, flow rates and solvent composition were investigated. Under suitable conditions, the DESI technique allows the preservation of the structural integrity of such fragile compounds. DESI enabled the sensitive detection of anionic hyaluronic acid and heparin oligosaccharides with a limit of detection (LOD) down to 5 fmol (≈10 pg) for the hyaluronic acid decasaccharide. Detection of hyaluronic acid oligosaccharides in urine sample was also successfully achieved with LOD values inferior to the ng range. Multistage tandem mass spectrometry (MSⁿ) through the combination of the DESI source with a hybrid linear ion trap‐orbitrap mass spectrometer allowed the discrimination of isomeric sulfated oligosaccharides and the sequence determination of a hyaluronic acid decasaccharide. These results open promising ways in glycomic and glycobiology fields where structure–activity relationships of bioactive carbohydrates are currently questioned. Copyright © 2012 John Wiley & Sons, Ltd.     

Combining desorption electrospray ionization mass spectrometry and nuclear magnetic resonance for differential metabolomics without sample preparation

Desorption electrospray ionization mass spectrometry (DESI-MS) and nuclear magnetic resonance (NMR) spectroscopy are used to provide data on urine examined without sample preparation to allow differentiation between diseased (lung cancer) and healthy mice. Principal component analysis (PCA) is used to shortlist compounds with potential for biomarker screening which are responsible for significant differences between control urine samples and samples from diseased animals. Similar PCA score plots have been achieved by DESI-MS and NMR, using a subset of common detected metabolites. The common compounds detected by DESI and NMR have the same changes in sign of their concentrations thereby indicating the usefulness of corroborative analytical methods. The effects of different solvents and surfaces on the DESI mass spectra are also evaluated and optimized. Over 80 different metabolites were successfully identified by DESI-MS and tandem mass spectrometry experiments, with no prior sample preparation.     

Molecular simulation of an amorphous poly(methyl methacrylate)–poly(tetrafluoroethylene) interface

Molecular dynamics calculations of an amorphous interfacial system of poly(methyl methacrylate) (PMMA) and poly(tetrafluoroethylene) (PTFE) containing about 10,000 interaction sites were performed for 15 ns under constant pressure and constant temperature conditions. The time evolutions of the thickness, density and number of atomic pairs in the interfaces suggested that the interfaces reached their equilibrium states with an interfacial thickness of about 2 nm at 500 K. The molecular motion in the interface and bulk was compared using mean square displacement and torsional autocorrelation function. The separation at a PMMA/PTFE interface was mimicked using non-equilibrium molecular dynamics calculations by applying the potential energy to the MD cell in a direction perpendicular to the interface. Initially, the PTFE layer close to the interface was deformed, and before complete separation, some segments of the PTFE molecules extended from the bulk to the surface of the PMMA layer, which were attached by the intermolecular interaction. The remaining PTFE molecules were entangled in the bulk, which probably prevented the transfer of the PTFE molecules to the surfaces of the PMMA layers. On the other hand, the PMMA layer was only slightly deformed. This separation behavior can be explained by taking into account the intermolecular interaction, the barrier to the conformational changes of the backbones and the entanglement of the PTFE molecules in the bulk.     

Scanning electron microscopic imaging of surface effects in desorption and nano-desorption electrospray ionization

Scanning electron microscopy was used to investigate rivulets that are formed on the analyzed surface during desorption electrospray ionization (DESI) experiment. Ferromagnetic nanoparticles added to the spray solvent in a form of colloid solution functioned as an additional surface probe. The existence of the rivulets was confirmed on glass and newly demonstrated on two different types of porous polytetrafluoroethylene (PTFE). The results show that in standard DESI set-up the rivulets are arranged into very regular shapes. Same rivulets were obtained in DESI experiments without high voltage on the sprayer. However, no such rivulets or any other regular patterns were found on a surface in nano-DESI (nanospray DESI without the carrier nebulizing gas) experiments. This indicates that symmetrical rivulets are created by the hydrodynamical rather than electrostatic forces. It was also demonstrated that blocking the rivulets by a simple physical barrier did not influence known surface charging effects.     

Dual silane surface functionalization for the selective attachment of human neuronal cells to porous silicon

Porous silicon (pSi) surfaces were chemically micropatterned through a combination of photolithography and surface silanization reactions. This patterning technique produces discretely defined regions on a pSi surface functionalized with a specific chemical functionality, and the surrounding surface displays a completely different functionality. The generated chemical patterns were characterized by a combination of IR microscopy and the conjugation of two different fluorescent organic dyes. Finally, the chemically patterned pSi surface was used to direct the attachment of neuronal cells to the surface. This patterning strategy will be useful for the development of high-throughput platforms for investigating cell behavior.     

Improved desorption electrospray ionization mass spectrometry performance using edge sampling and a rotational sample stage

The position of the surface to be analyzed relative to the sampling orifice or capillary into the mass spectrometer has been known to dramatically affect the observed signal levels in desorption electrospray ionization mass spectrometry (DESI‐MS). In analyses of sample spots on planar surfaces, DESI‐MS signal intensities as much as five times greater were routinely observed when the bottom of the sampling capillary was appropriately positioned beneath the surface plane (‘edge sampling’) compared with when the capillary just touched the surface. To take advantage of the optimum ‘edge sampling’ geometry and to maximize the number of samples that could be analyzed in this configuration, a rotational sample stage was integrated into a typical DESI‐MS setup. The rapid quantitative determination of caffeine in two diet sport drinks spiked with an isotopically labeled internal standard demonstrated the utility of this approach. Published in 2008 by John Wiley & Sons, Ltd.