Arkady I. Gusev

Effect of liquid chromatography separation of complex matrices on liquid chromatography-tandem mass spectrometry signal suppression.

The effect of liquid chromatography separation on liquid chromatography-tandem mass spectrometry (LC-MS-MS) signal response for the characterization of low-molecular-mass compounds in a complex matrix was investigated. Matrix induced signal suppression appears throughout the entire LC-MS-MS analysis of wheat forage extract, with greatest suppression occurring at early retention times. Experimental results show that co-elution of matrix components and analytes from the LC column may be most strongly attributed to column overloading rather than similar analyte and matrix retention behavior. As a result, two-dimensional (LC-LC) separation can be a highly effective approach to address signal suppression effects for the quantitative LC-MS-MS analysis of complex matrix samples.

Quantitative Analysis of Peptides by Matrix-Assisted Laser Desorption/Ionization Time-Of-Flight Mass Spectrometry

Quantitative analysis of peptides using internal standards was carried out with the use of matrix-assisted laser desorption/ionization (MALDI) on a time-of-flight mass spectrometer in a ferulic acid matrix. Spectra were collected and averaged over several areas of the sample surface at constant laser energy, and a defocused beam was used to improve reproducibility. A calibration curve was plotted, showing a correlation coefficient of 0.992 and a relative standard deviation of 1.83%. The accuracy and precision of this technique were sensitive to the crystal structure of the matrix and saturation effects of the instruments detector.

Characterization of polysiloxanes with different functional groups by time-of-flight secondary ion mass spectrometry.

Polydimethylsiloxane (PDMS), polyhydromethylsiloxane (PHMS), and polymethylphenylsiloxane (PMPhS) have been studied by TOF-SIMS to investigate effects of functional group changes on polymer fragmentation mechanisms. Cyclic fragments are observed in the low mass range spectra of PDMS and PHMS, but not in the spectrum of PMPhS. Effects of functional group substitution on the fragmentation mechanisms of polysiloxanes are evident in the high mass range spectra (>1000 Da). Peaks of oligomers cationized by silver dominate the high mass range of the spectra of all low molecular weight polysiloxanes. However, fragmentation patterns of these samples are different. Neutral cyclic fragments cationized by silver are identified in the high mass range of the spectra of PDMS and PHMS, but not in the spectrum of PMPhS. The major fragments of PHMS and PMPhS are [oligomer-14+Ag]+. The PHMS spectrum also shows peaks [oligomer-28+Ag]+. These distinctive fragmentation patterns can be used to identify the polysiloxanes.

Direct Quantitative Analysis from Thin-Layer Chromatography Plates Using Matrix-Assisted Laser Desorption/Ionization Mass Spectrometry

Direct quantitative analysis using thin-layer chromatography (TLC) coupled with matrix-assisted laser desorption/ionization mass spectrometry (MALDI) has been demonstrated. An internal standard and a data collection protocol were used for analysis directly from TLC plates to compensate for shot-to-shot signal degradation, as well as deviations of analyte and internal standard spatial distributions within the TLC spot. Cocaine hydrochloride was used as a model compound for this study, and cocaine-d3 was used as the internal standard. Quantitative analysis by TLC/MALDI yielded comparable results to those obtained with stainless steel substrates (the standard MALDI method) for point-to-point repeatability, % RSD of the standard curve, and measurement precision. For silica gel and reverse-phase TLC plates, the relative standard deviation of the standard curve slope was better than 3%, the relative standard deviations of the analyte/internal standard intensity ratios ranged from 3.8% to 9.5%, the precision was estimated to be better than 12%, and the detection limits were estimated to be 60 pg for both TLC plate types. Quantitative analysis using stainless steel substrates yielded a lower detection limit than that obtained by TLC/MALDI by a factor of six. Perspectives for improving the detection limits of direct TLC/MALDI quantitative analysis are discussed. Index Headings: MALDI; TLC; Quantification.

A novel interface for on-line coupling of liquid capillary chromatography with matrix-assisted laser desorption/ionization detection

A novel interface has been developed which should allow the direct on-line coupling of liquid capillary chromatography with matrix-assisted laser desorption/ionization (MALDI) mass spectrometry detection. The interface employs continuous analyte/matrix co-crystallization onto a porous frit installed at a capillary end which is used as the target for MALDI. After separation, the analyte effluent is premixed with the MALDI matrix solution and introduced into the interface. The analyte/matrix mixture is co-crystallized onto the frit surface in the vacuum environment of the mass spectrometer. Continuous matrix/analyte crystallization and interface regeneration is accomplished by a combination of solvent flushing and laser ablation. The memory effect is negligible over a dynamic range of ca. 200. Several applications, including analysis of small peptides and combination with gel permeation chromatography (GPC), have indicated that the on-line MALDI interface does not sacrifice chromatographic or mass spectral resolution, and have demonstrated the possibility of a reliable LC-MALDI system. Copyright 1999 John Wiley & Sons, Ltd.

Improvement of signal intensities in static secondary‐ion mass spectrometry using halide additives and substrate modification

A new approach is reported for secondary-ion time-of-flight mass spectrometry (TOF-SIMS) sample preparation. The method involves the use of halide additives or halide modification of silver substrate surfaces to promote analyte cationization and protonation. The enhancement of signal intensity has been demonstrated for neutral organic lipophilic and hydrophilic compounds including various small peptides and nucleosides. Improvement factors range from 2–30 for cationized species to 20–2000 for protonated species. However, the new sample preparation does not affect the signal intensities of preformed ionic species. The sample preparation approach is applicable to a wide variety of neutral compounds and should find broad use for organic analysis by TOF-SIMS.

A quantitative study of in vitro hepatic metabolism of tacrolimus (FK506) using secondary ion and matrix-assisted laser desorption/ionization mass spectrometry.

The identification and simultaneous quantification of Tacrolimus and its hepatic metabolites in baboons has been achieved using matrix-assisted laser desorption/ionization (MALDI) time-of-flight (TOF) mass spectrometry and static secondary-ion mass spectrometry (TOF-SIMS). Little fragmentation, high sensitivity and tolerance to contamination are the major advantages of these methods, allowing facile identification and quantification of metabolites produced in vitro with minor analyte isolation. Based on the MALDI and TOF-SIMS results, seven metabolites have been identified: de-methylated, di de-methylated, hydroxylated, di hydroxylated, de-methylated hydroxylated, dihydrodiol, and di de-methylated hydroxylated. The concentrations of the parent drug and its major metabolites (e.g. de-methylated, di de-methylated) were measured using Rapamycin as an internal standard. The time course of Tacrolimus and its major metabolites as a function of incubation time was calculated. Good correlation between SIMS and MALDI results was obtained.

Analysis of Cationic Pesticides by Thin Layer Chromatography/Matrix — Assisted Laser Desorption Ionization Mass Spectrometry

Abstract This study demonstrates the analysis of pesticides by direct coupling of Thin Layer Chromatography (TLC) with Matrix-Assisted Laser Desorption Ionization mass spectrometry (MALDI). TLC-MALDI takes advantage of the high sensitivity of MALDI while utilizing the relatively quick and inexpensive separation offered by TLC. The optimization of a protocol to analyze cationic pesticides by TLC-MALDI is reported and tested on normal phase, reverse phase, and cellulose TLC plates. Detection limits in the picogram range were found for two analytes, phosphon and avenge. Detection limits for glyodin were in the nanogram range. Consistency in detection limits for each compound is observed on all types of stationary phases investigated. This demonstrates the applicability of this method for use on a variety of TLC plates.

Species-dependent hepatic metabolism of immunosuppressive agent tacrolimus (FK-506).

The current study aims to investigate species-related differences in the in-vitro hepatic metabolism of tacroliums using liver microsomes obtained from rat, hamster, guinea pig, rabbit, pig, dog, baboon and humans. Tacrolimus metabolism was characterized using high-performance liquid chromatography- ultraviolet light (HPLC-UV) and two soft ionization mass spectrometric techniques; matrix-assisted lasers desorption/ionization (MALDI) and time-of-flight-secondary ion mass spectrometry (TOF-SIMS). The extent of tacrolimus metabolism, when normalized to the cytochrome P-450 content, was in the order: rat < hamster < rabbit < pig < guinea pig < dog < human < baboon. Tacrolimus metabolism exhibited significant qualitative and quantitative differences between the animal species tested. Desmethyl- (MI–MIII), didesmethyl- (MIV–MVI), monohydroxy- (MVII), dihydroxy- (MVIII), epoxide- (MIX), dihydrodiol- (MX), monodesmethyl and monohydroxy- (MXI–MXIII), and didesmethyl and monohydroxy- (MXIV–MXVI) tacroliums metabolites were identified in the species tested. MI–MX were identified in all the species tested; MXI–MXVI were identified in all species except rat, rabbit and guinea pig; and MXIV–MXVI were identified only in baboon. The current investigation was unable to detect any phase II metabolites due to the limitations of the test system used. The analytical methods were not able to differentiate optical and positional isomers of metabolites due to the nature of the analytical tools used, therefore groups of metabolites were identified based on their molecular weights and available information. From the current in-vitro metabolism studies, the pattern of tacroliums metabolism in baboons closely resembled that in humans and thus it is ideal for studying tacroliums metabolism-related work of clinical relevance.

Stereoregular polypropylenes studied by time‐of‐flight secondary ion mass spectrometry (ToF‐SIMS) and atomic force microscopy (AFM)

Time-of-flight secondary ion mass spectrometry (ToF-SIMS) and atomic force microscopy (AFM) were utilized to characterize polypropylenes of different stereoregularity (atactic, syndiotactic and isotactic) deposited on gold- or silver-coated mica surfaces. The AFM images clearly revealed distinctive surface topographies among the three types of polymers, which are largely due to their stereoregularity and crystallinity. The ToF-SIMS data showed distinguishable differences among the fragmentation spectra obtained from these polypropylene samples, especially in the low-mass region. Variations in the cluster structures were also detected. Supporting metal substrates had significant influence on fragment ion formation. Our results suggest that mass spectrometry and AFM could be used together to obtain supplemental information to NMR measurements of polymer stereoregularity. Copyright