Sensitive Mass Spectrometer Research Articles

Comparison of Various Dilutions and Solid-Phase Extraction Cleanup on the Determination of Ephedrine-Type Alkaloids and Internal Standard Recovery in Ephedra Botanical Raw Material and Powdered Extract

A study was conducted to determine the effect of 3 dilution levels on the precision of the ephedra alkaloid method when used in conjunction with a solid-phase extraction (SPE) column. For the dilutions studied, SPE column cleanup is necessary because it promotes a greater recovery of the internal standard. However, overall, target precision values were not obtained on the test materials. It was determined that the SPE column is not the cause of the lower recovery in the more concentrated solutions. Significant signal suppression of the internal standard occurs in more concentrated solutions within the mass spectrometer. It is hypothesized that this lack of performance on the part of the SPE column may be linked to its inability to fully clean contaminants from the higher concentration solutions and/or a mass spectrometer overload, which resulted in the internal standard not fully correcting for signal suppression in more concentrated solutions. An internal standard is necessary, especially for accuracy, for the determination of all alkaloids, and only dilute solutions can be accurately analyzed. Due to the sensitivity of the mass spectrometer, it is recommended that the determination of ephedrine alkaloids in dietary supplements and botanicals should be studied using a standard graph at a level 10 x less than the current method. It is also recommended that the SPE column used should be evaluated on its need for the sample dilutions made to fit the newly recommended standard graph.

Novel Evaluation System for Extreme Ultraviolet Lithography Resist in NewSUBARU

A standard assessment system for resist development with extreme ultraviolet (EUV) light was developed. The system for the evaluation of an EUV resist was installed at the BL3 beamline in the NewSUBARU synchrotron radiation facility. This optical system simulates a six-mirror imaging system. Incident light is reflected seven times in the chamber. The centroid wavelength is 13.57 nm. The beam size at the sample surface is 4×4 mm2 and we can take twelve shots for each sample by moving the sample in the lateral direction. Furthermore, a highly sensitive quadrupole mass spectrometer that can measure the mass numbers of ion species from 1 to 500 and is connected to a resist evaluation chamber is used for resist outgassing analysis under EUV irradiation. The main purpose of this whole system is to evaluate the basic physical and chemical properties of various EUV resists candidates, such as sensitivity and outgas characteristics.

Single run measurements of drug‐protein binding by high‐performance frontal analysis capillary electrophoresis and mass spectrometry

A novel drug-protein binding measurement method based on high-performance frontal analysis and capillary electrophoresis (HPFA/CE) is presented. A single run measurement approach is proposed to circumvent utilization of a calibration curve that is often performed with HPFA. A sensitive mass spectrometer is applied as a detector enabling the measurement of in vitro protein binding at lower drug concentrations. Unbound free fraction and binding constants can be determined by a single run measurement by consecutive injections of an internal drug standard, a buffer plug and a drug-protein mixture. Effects of injection volumes on peak height and plateau profile were investigated in two different separation systems, non-volatile buffer and volatile buffer, with UV and mass spectrometry detection, respectively. A simplified one-to-one binding model is employed to evaluate the proposed method by using both single and multiple drug concentrations to measure the unbound free fraction and calculate the binding constants of some selected compounds. The method is suitable for rapid and direct screening of the binding of a drug to a specific protein or drug-plasma protein binding.

Protein Stoichiometry of a Multiprotein Complex, the Human Spliceosomal U1 Small Nuclear Ribonucleoprotein: ABSOLUTE QUANTIFICATION USING ISOTOPE-CODED TAGS AND MASS SPECTROMETRY

The human U1 snRNP (small nuclear ribonucleoprotein), which is a part of the spliceosome, consists of U1 snRNA and ten different proteins: seven Sm proteins B/B', D1, D2, D3, E, F, and G and the three U1-specific proteins U1-70 K, U1-A, U1-C. To determine the stoichiometry of all ten proteins, the complex was denatured, digested completely with an endoproteinase and labeled with an amine-specific tag. Corresponding peptides were synthesized and labeled with the same tag containing heavier isotopes. The digest was then spiked with defined amounts of the synthetic peptides, and the resulting isotopic peptide pairs were analyzed quantitatively by mass spectrometry. The mass spectra provided information about the absolute amount of each component in the starting protein mixture. The use of the isotope-coded, amine-specific reagents propionyl-N-oxysuccinimide and nicotinoyl-N-oxysuccinimide was evaluated for stoichiometry determination; the nicotinoyl reagent was found to be advantageous because of its greater mass spectrometric sensitivity. Absolute quantities of all ten proteins were measured, showing equal numbers of all ten proteins in the U1 spliceosomal snRNP. These data demonstrate that quantitative mass spectrometry has great potential for the determination of the stoichiometry of multiprotein complexes.

Liquid chromatography‐mass spectrometry for the quantitative bioanalysis of anticancer drugs

The monitoring of anticancer drugs in biological fluids and tissues is important during both pre-clinical and clinical development and often in routine clinical use. Traditionally, liquid chromatography (LC) in combination with ultraviolet (UV), fluorescence, or electrochemical detection is employed for this purpose. The successful hyphenation of LC and mass spectrometry (MS), however, has dramatically changed this. MS detection provides better sensitivity and selectivity than UV detection and, in addition, is applicable to a significantly larger group of compounds than fluorescence or electrochemical detection. Therefore, LC-MS has now become the method of first choice for the quantitative bioanalysis of many anticancer agents. There are still, however, a lot of new developments to be expected in this area, such as the introduction of more sensitive and robust mass spectrometers, high-throughput analyses, and further optimization of the coupled LC systems. Many articles have appeared in this field in recent years and are reviewed here. We conclude that LC-MS is an extremely powerful tool for the quantitative analysis of anticancer drugs in biological samples.

Zooming in: Fractionation strategies in proteomics

The recent development of mass spectrometry, i.e., high sensitivity, automation of protein identification and some post-translational modifications (PTMs) significantly increased the number of large-scale proteomics projects. However, there are still considerable limitations as none of the currently available proteomics techniques allows the analysis of an entire proteome in a single step procedure. On the other hand, there are several successful studies analyzing well defined groups of proteins, e.g., proteins of purified organelles, membrane microdomains or isolated proteins with certain PTMs. Coupling of advanced separation methodologies (different prefractionation strategies, such as subcellular fractionation, affinity purification, fractionation of proteins and peptides according to their physicochemical properties) to highly sensitive mass spectrometers provides powerful means to detect and analyze dynamic changes of low abundant regulatory proteins in eukaryotic cells on the subcellular level. This review summarizes and discusses recent strategies in proteomics approaches where different fractionation strategies were successfully applied.

Determination of a neuroprotective agent ( S)-(+)-BMS-204352 in human, rat and dog plasma by enantioselective liquid chromatography-tandem mass spectrometry

A sensitive liquid chromatography-electrospray ionization tandem mass spectrometry method (LC/ESI/MS/MS) for the enantioselective determination of ( S)-(+)-BMS-204352, a potent and specific maxi-K channel opener, in human, rat and dog plasma was developed. ( S)-(+)-BMS-204352, its enantiomer ( R)-(−)-BMS-204353 and the internal standard ( 13C-deuterated racemate of ( S)-(+)-BMS-204352) were extracted from plasma using toluene. Chromatographic separation for the enantiomers was achieved on a Chiralcel OD-H analytical column with a run time of 8 min. An aqueous mobile phase modifier was added post column to enhance the mass spectrometer sensitivity. ESI mass spectra were acquired in the negative mode with selected reaction monitoring. The limit of quantitation (LLOQ) is 0.10 ng/mL for human plasma assay. Samples from a clinical study and two animal studies were processed using these procedures. Based on the in vivo data, lack of inversion of ( S)-(+)-BMS-204352 to ( R)-(−)-BMS-204353 was demonstrated in human, rat and dog after administration of the drug. A sensitive non-enantioselective LC/ESI/MS/MS assay has also been developed for ( S)-(+)-BMS-204352 which uses a similar extraction procedure with a C18 column with a limit of quantitation at 0.05 ng/mL. Human study samples were analyzed by both methods and the correlation coefficient between both sets of data is greater than 0.99.

Design and operational characteristics of a cast steel mass spectrometer

A cast steel magnetic sector mass analyzer is developed for studies of hydrogen and helium ion beams generated by a gas discharge compact ion source. The optimum induced magnetic flux density of 3500 G made it possible to scan the whole spectrum of hydrogen and helium ion species. Analysis of beam characteristics shows that the mass spectrometer sensitivity, and resolving power are approximately inversely proportional. The resolution is enhanced at higher pressures and lower current discharges. In contrast, the instrument sensitivity increased at higher current discharges and decreased at higher pressures. Calculations of the ultimate resolving power with reference to analyzer dimensions yield a numerical value of 30. System anomaly in the form of spherical aberrations was also analyzed using the paraxial beam envelope equation. Beam divergence is most significant at high discharge conditions where angular spread reaches an upper limit of 8.6°.

Charge competition and the linear dynamic range of detection in electrospray ionization mass spectrometry

An experimental investigation and theoretical analysis are reported on charge competition in electrospray ionization (ESI) and its effects on the linear dynamic range of ESI mass spectrometric (MS) measurements. The experiments confirmed the expected increase of MS sensitivities as the ESI flow rate decreases. However, different compounds show somewhat different mass spectral peak intensities even at the lowest flow rates, at the same concentration and electrospray operating conditions. MS response for each compound solution shows good linearity at lower concentrations and levels off at high concentration, consistent with analyte “saturation” in the ESI process. The extent of charge competition leading to saturation in the ESI process is consistent with the relative magnitude of excess charge in the electrospray compared to the total number of analyte molecules in the solution. This ESI capacity model allows one to predict the sample concentration limits for charge competition and the on-set of ionization suppression effects, as well as the linear dynamic range for ESI-MS. The implications for quantitative MS analysis and possibilities for effectively extending the dynamic range of ESI measurements are discussed.

Analysis of serum proteomic patterns for early cancer diagnosis: drawing attention to potential problems.

In a recent update (1) of already impressive data (2), it was reported that the use of proteomic patterns in serum to diagnose ovarian and prostate cancers can achieve perfect diagnostic sensitivity and specificity. A diagnostic sensitivity and specificity of 100% is unprecedented for any tumor marker known to date and, if reproducible, this finding could have a major impact on the way we diagnose cancer in the future. Over the last 2 years, results reported by several groups (2– 6) have suggested that such proteomic patterns, particularly those generated by surface-enhanced laser desorption/ionization time-of-flight (SELDI-TOF) mass spectrometry, may facilitate the early diagnosis of various cancers, including those of the ovary, prostate, breast, and bladder. SELDI-TOF proteomic profiling technology has been reviewed (7,8). The impressive results reported with this new technology were welcomed by scientists, the popular press, the public, and even by politicians (9). Although there has been little published criticism of this methodology (10–12), serious skepticism about its utility has been expressed publicly at various scientific meetings. Many investigators and clinicians have adopted a wait-andsee approach pending the outcomes of prospective clinical studies using this technology which are starting now but will require years to complete. Here, I summarize some shortcomings of this technology for the purpose of stimulating further discussion and research. Considering what is known about tumor markers, the mechanisms by which they are released into the circulation, their abundance in biologic fluids, their metabolism and excretion, and their dynamic relationships within the host, it is unlikely that proteomic profiling by SELDI-TOF methods will be a useful approach for the diagnosis of cancer. Moreover, it is conceivable that published data may, in fact, be biased by artifacts related to the nature of the clinical samples used, the mass spectrometry instrument, and/or the bioinformatic analysis. In a recent meta-analysis (12) of prostate cancer proteomic data from four papers by three different research groups, I pointed out that the discriminatory peaks (i.e., peaks representing molecules that appear or disappear during cancer progression, or whose amounts differ in cancerous versus noncancerous tissue) identified in the four papers were very different, even in the two papers published by the same group using the same experimental data but different bioinformatic tools (12,13). These data are summarized in Table 1. These discrepancies suggest that serum proteomic patterns obtained by the SELDITOF technique may not be reproducible and that the discriminatory peaks are not consistent either within a group or among groups of investigators for the same type of cancer, even when the general analytical methods or datasets are the same. Furthermore, the reported diagnostic sensitivities and specificities of prostate cancer diagnosis based on SELDI-TOF technology differ substantially among the four reports. Another rather surprising phenomenon associated with these data is that serum proteins that are known to distinguish patients with benign conditions from patients with malignancies (e.g., prostatespecific antigen in prostate cancer) were not identified by this new technology, raising serious questions about its analytic sensitivity. After reviewing the serum levels of known tumor markers for various malignancies and the current sensitivity of mass spectrometers, I have concluded that the SELDI-TOF technology that is currently used for serum analysis is not capable of detecting any serum component at concentrations of less than 1 g/mL (12). This range of concentrations is approximately 1000-fold higher than the concentrations of known tumor markers in the circulation (12). This analysis led me to conclude that the serum discriminatory peaks identified by mass spectrometry very likely represent high-abundance molecules that were unlikely to have been released into the circulation by very small tumors or their microenvironments. I suggested that the discriminatory peaks may instead represent acute-phase reactants (i.e., molecules whose serum concentrations are increased in patients with acute or chronic inflammatory conditions) or other proteins or protein fragments that are released into the circulation by large organs, such as the liver, in response to the presence of a tumor or cancer epiphenomena, such as infection, inflammation, or malnutrition (10,11). Alternatively, some of these proteomic changes may represent artifacts of sample collection, storage, or pretreatment, patient selection, or other idiosyncrasies. Little effort has been made to positively identify at least some of the molecules that comprise the discriminatory peaks to understand their origins and why their levels are altered. Indeed, in the few cases where such peaks were identified, some were composed of high-abundance molecules released by the liver and others were composed of acute-phase reactants (Table 2) (16 –18). At the 2003 annual meeting of the American Association for Cancer Research, Zhang et al. (16) reported the identities of three discriminatory peaks in ovarian cancer: apolipoprotein A1, transthyretin (pre-albumin) fragment, and inter-alpha-trypsin inhibitor. Others identified haptoglobinsubunit for ovarian cancer (17) and vitamin

Quantitative analysis of squalamine, a self-ionization-suppressing aminosterol sulfate, in human plasma by LC–MS/MS

The special physico-chemical property of squalamine enables the formation of intra- or inter-molecular non-volatile strong salt, which is difficult to ionize in a mass spectrometer’s interface. A sensitive, accurate, precise, and specific method for the quantitative determination of this self ion-suppressing compound in human plasma has been developed and validated using high performance liquid chromatography (HPLC) coupled with positive electrospray tandem mass spectrometry (MS/MS). Solid phase extraction (SPE) technique was utilized to extract human plasma samples using the Waters Oasis HLB cartridges. Deuterated squalamine was used as the internal standard (IS). Positive multiple reaction monitoring (MRM) mode was used to achieve both sensitivity and selectivity. A quadratic linearity range over 5–1000 ng/ml, R>0.999 was achieved. Performance of the method has been validated and met all the specifications set forth in the US Food and Drug Administration’s May 2001 “Bioanalytical Method Validation Guidance for Industry”. Different sample reconstitution solutions were found to have dramatic impact on sensitivity of mass spectrometer used to squalamine. This is the first quantitation method using a positive and true multiple reaction monitoring mode detection for squalamine.

Valve packing tests leakage measurement equipment sensitivity

During third-party tests on James Walker’s Supagraf Premier valve stem packing, emission levels round the valve stem were so low that they challenged the sensitivity of the helium mass spectrometer used to measure the leak rate. This is a short news story only. Visit www.worldpumps.com for the latest pump industry news.

Proteomics of the Chloroplast Envelope Membranes from Arabidopsis thaliana

The development of chloroplasts and the integration of their function within a plant cell rely on the presence of a complex biochemical machinery located within their limiting envelope membranes. To provide the most exhaustive view of the protein repertoire of chloroplast envelope membranes, we analyzed this membrane system using proteomics. To this purpose, we first developed a procedure to prepare highly purified envelope membranes from Arabidopsis chloroplasts. We then extracted envelope proteins using different methods, i.e. chloroform/methanol extraction and alkaline or saline treatments, in order to retrieve as many proteins as possible, from the most to least hydrophobic ones. Liquid chromatography tandem mass spectrometry analyses were then performed on each envelope membrane subfraction, leading to the identification of more than 100 proteins. About 80% of the identified proteins are known to be, or are very likely, located in the chloroplast envelope. The validation of localization in the envelope of two phosphate transporters exemplifies the need for a combination of strategies to perform the most exhaustive identification of genuine chloroplast envelope proteins. Interestingly, some of the identified proteins are found to be Nalpha-acetylated, which indicates the accurate location of the N terminus of the corresponding mature protein. With regard to function, more than 50% of the identified proteins have functions known or very likely to be associated with the chloroplast envelope. These proteins are a) involved in ion and metabolite transport, b) components of the protein import machinery, and c) involved in chloroplast lipid metabolism. Some soluble proteins, like proteases, proteins involved in carbon metabolism, or proteins involved in responses to oxidative stress, were associated with envelope membranes. Almost one-third of the proteins we identified have no known function. The present work helps understanding chloroplast envelope metabolism at the molecular level and provides a new overview of the biochemical machinery of the chloroplast envelope membranes.

Measuring Electrocatalytic Activity on a Local Scale with Scanning Differential Electrochemical Mass Spectrometry

This paper describes a new technique entitled scanning differential electrochemical mass spectrometry (SDEMS) that combines a quadrupole mass spectrometer with a membrane-covered capillary inlet and a high resolution positioning system that is designed to perform spatial mapping in solution near an electrode interface. Potential applications of this technique include the local characterization of anode catalysts for fuel cells as well as a range of analytical measurements and combinatorial screening studies. The capabilities of this technique are demonstrated by monitoring product evolution in several model electrocatalytic reactions, including the hydrogen evolution reaction, carbon monoxide oxidation, and the direct oxidation of methanol on platinum and platinum-ruthenium electrodes. The inlet of the SDEMS is based upon a small diameter capillary tube to which a nanoporous, hydrophobic membrane is attached. The capillary inlet is positioned near a substrate electrode using a three-dimensional positioning system. The effect of capillary substrate separation and substrate current on the sensitivity and time response of mass spectrometer’s ion current are illustrated during hydrogen evolution at a platinum substrate. The sensitivity is demonstrated further by detection of carbon dioxide evolution during the oxidation of a monolayer of carbon monoxide adsorbed on platinum. The ability to address more complex reactions involving complete and partial oxidation products is illustrated with methanol oxidation. In order to demonstrate the ability of this technique to perform spatial mapping, an eight-element band electrode was interrogated for hydrogen evolution and methanol oxidation. Detection of ion currents associated with complete and partial oxidation products of methanol on a set of platinum-ruthenium band electrodes illustrates the ability of this method to spatially discriminate between various reactive sites on a surface, which has potential utility in analytical characterization as well as application as a screening tool in combinatorial catalysis studies. © 2003 The Electrochemical Society. All rights reserved.

Analysis of native and chemically modified oligonucleotides by tandem ion‐pair reversed‐phase high‐performance liquid chromatography/electrospray ionization mass spectrometry

Ion-pair reversed-phase high-performance liquid chromatography (IP-RP-HPLC) was utilized in tandem with negative-ion electrospray ionization time-of-flight mass spectrometry (ESI-TOFMS) for the analysis of native and chemically modified oligonucleotides. Separation was performed on a 1.0 x 50 mm column packed with porous C(18) sorbent with a particle size of 2.5 microm and an average pore diameter of 140 A. A method was developed which maximizes both chromatographic separation and mass spectrometric sensitivity using an optimized buffer system containing triethylamine and 1,1,1,3,3,3-hexafluoro-2-propanol with a methanol gradient. The ESI-TOFMS tuning parameters were also optimized in order to minimize in-source fragmentation and achieve the best sensitivity. Analyses of native, phosphorothioate, and guanine-rich oligonucleotides were performed by LC/MS. Detection limits were at sub-picomole levels with an average mass accuracy of 125 ppm. The described method allowed for the LC/MS analysis of oligonucleotides up to 110mer in length with little alkali cation adduction. Since sensitive detection of oligonucleotides was achieved with ultraviolet (UV) detection, we utilized a combination of UV-MS for quantitation (UV) and characterization (MS) of oligonucleotides and their failure sequence fragments/metabolites.

Analysis of low-molecular-mass organic acids using capillary zone electrophoresis–electrospray ionization mass spectrometry

A capillary zone electrophoresis–electrospray ionization-mass spectrometry (CZE–ESI-MS) method was developed to facilitate identification and determination of eleven low-molecular-mass (LMW) organic acids (i.e. oxalic, lactic, malonic, maleic, citric, tartaric, adipic, glutaric, gluconic, isosaccharinic and succinic acid) in different sample matrices. This CZE method was adapted to suit MS conditions. Sheath liquid, sheath flow and MS parameters were optimized to achieve high mass spectrometric sensitivity. The CZE–ESI-MS procedure showed good sensitivity (limit of detection of <0.05–0.7 mg/l for all acids), linearity ( r 2=0.9925–0.9998) and reproducibility (2.09–5.34% RSD). The applicability of the CZE–ESI-MS was demonstrated on LMW organic acids in an ale sample. In addition the (here presented) method also provided quantification of fumaric, galacturonic and 2-ketoglutaric acid with high sensitivity.

Zircon U-Th-Pb Geochronology by Isotope Dilution -- Thermal Ionization Mass Spectrometry (ID-TIMS)

ID-TIMS is the acronym for Isotope Dilution Thermal Ionization Mass Spectrometry. This refers to the addition of an isotope tracer to a dissolved sample to make a homogeneous isotopic mixture, and the measurement of isotopic composition of the mixture using a thermal ionization mass spectrometer. The method is one of the most accurate and precise methods of isotopic techniques because it is relatively insensitive to chemical yields or mass spectrometric sensitivity. It is a method very widely applied both in earth and many other areas of science involving the measurement of element or isotope concentrations and isotopic ratios. The ID-TIMS technique was first applied to the U-Th-Pb dating of zircon in the 1950s (Tilton et al. 1955, Wetherill 1956, Tilton et al. 1957), exploiting the general availability to academia of enriched uranium isotopes developed in the 1940s and 1950s related to nuclear energy research. ID-TIMS has remained the main foundation to zircon geochronology ever since, in spite of the proliferation of other analytical methodologies. In the past 50 years, many improvements have been made and they have contributed to the maturity and reliability of the method. As a method, it was effectively unchallenged until the 1980s when secondary ionization mass spectrometry (Anderson and Hinthorne 1972) was further developed and applied to zircon geochronology by W. Compston and colleagues at the Australian National University (Compston et al. 1984). The instrument developed by the ANU group (SHRIMP, or Sensitive High Resolution Ion Microprobe) and the associated measurement protocols facilitated measurement of Pb/U isotopic ratios within a small region of a single zircon grain, and it proved to be a powerful tool to address complex age structure of multi-component zircons. In the 1990s, laser ablation quadrupole ICP-MS methods came on stream and offered an alternate way to make intra-grain U-Th-Pb isotopic …

Determination of 129I/127I isotope ratios in liquid solutions and environmental soil samples by ICP-MS with hexapole collision cell

The determination of 129I in environmental samples at ultratrace levels is very difficult by ICP-MS due to a high noise caused by Xe impurities in argon plasma gas (interference of 129Xe+), possible 127IH2+ interference and an insufficient abundance ratio sensitivity of the ICP mass spectrometer for 129I/127I isotope ratio measurement. A sensitive, powerful and fast analytical technique for iodine isotope ratio measurements in aqueous solutions and contaminated soil samples directly without sample preparation using ICP-MS with a hexapole collision cell (ICP-CC-QMS) was developed. Oxygen is used as reaction and carrier gas for iodine thermal desorption via the gas phase from solid environmental material in the sample introduction device coupled on-line to ICP-CC-QMS. A mixture of oxygen and helium as reaction gases in the hexapole collision cell was applied for reducing disturbing background intensity of 129Xe+. After optimization of measurement procedures the detection limit for 129I+ in aqueous solution was 8 × 10−13 g ml−1, which is better by about two orders of magnitude in comparison to the detection limit for 129I+ in sector field ICP-MS. The detection limit for direct 129I+ determination in contaminated environmental (soil) samples via gas-phase desorption without any additional sample preparation was 3 × 10−11 g g−1 (30 ppt). Furthermore, the results of the determination of 129I/127I isotope ratios at the 10−5–10−6 level in synthetic laboratory standards and environmental soil samples from contaminated areas are given.

Comparison of peak shapes obtained with volatile (mass spectrometry-compatible) buffers and conventional buffers in reversed-phase high-performance liquid chromatography of bases on particulate and monolithic columns

Retention factor, column efficiency and asymmetry factor were recorded for nine basic compounds on a number of RP-HPLC columns using phosphate and a variety of (MS-compatible) volatile mobile phase buffers of acid and neutral pH, in order to assess any effects of the buffer on performance. With formic or acetic acid, some phases gave partial or complete solute exclusion effects (reduced or negative k) compared with results using phosphate buffers at low pH. Despite its possible suppression of mass spectrometer sensitivity, trifluoroacetic acid was useful in enhancing retention times of relatively hydrophilic protonated bases, due to ion-pair effects. Peak shape was relatively poor on some pure silica-based ODS phases at pH 7 compared with results at acid pH. At low pH and at pH 7, ammonium and potassium phosphate gave very similar k, but the former may be preferable due to its volatile cation. Improved peak shapes, attributed to superior silanol masking effects, were obtained with ammonium phosphate at pH 7, but not at acid pH. Ammonium acetate gave acceptable peak shape at pH 7, but due to very limited buffer capacity, poor results were obtained for solutes having a p K a close to the mobile phase pH. Due to its instability, ammonium hydrogen carbonate is not a viable alternative buffer at pH 7.

On-line overpressure thin-layer chromatographic separation and electrospray mass spectrometric detection of glycolipids.

On-line thin-layer chromatographic separation and electrospray mass spectrometry (TLC/ESI-MS) has been accomplished by direct linking of a commercial overpressure TLC instrument, OPLC 50, and a Q-TOF mass spectrometer. Mass spectrometric detection sensitivity and chromatographic resolution achieved by this configuration were assessed using acidic glycolipids as examples. Under the optimized conditions, a sensitivity of 5 pmol of glycosphingolipid was readily demonstrated for TLC/ESI-MS and 20 pmol for TLC/ESI-MS/MS production scanning to derive the saccharide sequence and long chain base/fatty acid composition of the ceramide. Initial preconditioning of TLC plates is necessary to achieve high sensitivity detection by reducing chemical background noise. Plates can be used repeatedly (at least 10 times) for analysis, although this may result in a minor reduction in TLC resolution. Following solvent development, separated components on the TLC plates can be detected in the conventional way by nondestructive staining or UV absorption or fluorescence and can be stored for on-line TLC/ESI-MS analysis at a later stage without reduction in mass spectrometric detection sensitivity and chromatographic resolution. Aspects for further improvement of OPLC instrumentation include use of narrower TLC plate dimensions and refined design of the eluate exit system.