Heart-cutting Approach Research Articles

Boosting the odour perception of trace compounds exploiting three-dimensional GC-O with MS/FID simultaneous detection

ABSTRACT The study describes the effectiveness of a 3D GC system coupled with olfactometric detection and simultaneous MS and FID (3DGC-O-MS/FID) to improve the detection of odorants. Mega-bore columns were used to ensure a sample capacity able to enhance the odour perception. The heart-cut approach allowed to overcome the low efficiency of wide-bore columns, granting to purify enriched target fractions before olfactometry detection. After the third GC dimension, the isolated target peaks were eluted to the olfactometric port allowing the odour to last multiple seconds, making the panelist’s perception easier and more comfortable. The simultaneous FID/MS detection after the third GC dimension allowed to match specific odours to target molecules and their concentration in the sample. The 3DGC-O-MS/FID system boosts odour perception of trace components compared to conventional GC-O approaches. A reduced detection threshold of mg/kg levels was obtained compared to the mg/kg levels typical of monodimensional GC-O.

Profiling of sphingolipids in Caenorhabditis elegans by two-dimensional multiple heart-cut liquid chromatography – mass spectrometry

Sphingolipids exert important functions in cells, ranging from stabilising the cell membrane to bioactive signalling in signal transduction pathways. Changed concentrations of sphingolipids are associated with, among others, neurodegenerative and cardiovascular diseases. In this work, we present a novel two-dimensional liquid chromatography method (2D-LC) coupled to tandem mass spectrometry (MS/MS) for the identification of ceramides, hexosylceramides and sphingomyelins in the model organism Caenorhabditis elegans (C. elegans). The method utilises a multiple heart-cut approach with a hydrophilic interaction liquid chromatography (HILIC) separation in the first dimension. The fractions of the sphingolipid classes were cut out and thereby separated from the abundant glycerolipids, which offers a simplified sample preparation and a high degree of automation as it compensates the alkaline depletion step usually conducted prior to the chromatographic analysis. The fractions were stored in a sample loop and transferred onto the second column with the combination of two six port valves. A reversed phase liquid chromatography was performed as the second dimension and allowed for a separation of the species within a sphingolipid class and according to the fatty acid moiety of the sphingolipid. The segregation of the abundant glycerolipids and the reduced matrix effects allowed for better identification of low abundant species, especially dihydro-sphingolipids with a saturated sphingoid base. In addition, the separation of the three fractions was carried out parallel to the separation and equilibration in the first dimension, which leads to no extension of the analysis time for the 2D-LC compared to the one-dimensional HILIC method. In total 45 sphingolipids were detected in the C. elegans lipid extract and identified via accurate mass and MS/MS fragments.

Mass spectrometric investigation of cardiolipins and their oxidation products after two-dimensional heart-cut liquid chromatography

The anionic phospholipid class of cardiolipins (CL) is increasingly attracting scientific attention in the recent years. CL can be found as a functional component of mitochondrial membranes in almost all living organisms. Changes in the CL composition are favored by oxidative stress. Based on this finding, the investigation of CL and their oxidation products in relation to various disease patterns, including neurodegenerative ones, is moving into the focus of current research. The analysis of this diverse lipid class is still challenging and requires sensitive and selective methods. In this work, we demonstrate an online two-dimensional liquid chromatography (2D-LC) approach by means of a heart-cut setup. In the first dimension, a fast hydrophilic interaction liquid chromatography (HILIC) method was developed for the separation of CL and their oxidation products from other phospholipid classes, but more important from nonpolar lipid classes, such as triacylglycerol and cholesterol. Those classes can negatively affect the electrospray ionization and also the chromatography. For the heart-cut approach, the CL fraction was selectively transferred to a loop using a six-port valve followed by the transfer to a reversed phase (RP) column in second dimension. On the RP column, the transferred CL fraction including the oxidation products were separated according to the hydrophobicity of acyl chain moieties. Matrix effects were significantly reduced compared to the one-dimensional LC-MS method. In addition, the total separation time had not to be prolonged by shifting the equilibration step of the RP column parallel to the separation in first dimension. The heart-cut LC-LC approach was applied to artificially oxidized lipid extracts of bovine heart and yeast by means of Fenton reaction. In summary, 42 species have been identified by high resolution mass spectrometry and database matching. 31 species thereof have been further characterized by MS/MS experiments.

Interference-free mass spectrometric detection of capillary isoelectric focused proteins, including charge variants of a model monoclonal antibody.

CIEF represents an elegant technique especially for the separation of structural similar analytes, whereas MS is a state-of-the-art instrumentation for the identification and characterization of biomolecules. The combination of both techniques can be realized by hyphenating CIEF with CZE-ESI-MS applying a mechanical valve. During the CZE step, the remaining ESI-interfering components of the CIEF electrolyte are separated from the analytes prior to MS detection. In this work, a multiple heart-cut approach is presented expanding our previous single heart-cut concept resulting in a dramatical reduction of analysis time. Moreover, different sample transfer loop volumes are systematically compared and discussed in regard to peak width and transfer efficiency. With this major enhancement, model proteins (1.63-9.75mg/L), covering a wide pI range (5-10), and charge variants from a deglycosylated model antibody were analyzed on intact level. The promising CIEF-CZE-MS setup is expected to be applicable in different bioanalytical fields, e.g. for the fast and information rich characterization of therapeutic antibodies.

Quantification of ascorbic acid and acetylsalicylic acid in effervescent tablets by CZE-UV and identification of related degradation products by heart-cut CZE-CZE-MS.

Capillary electrophoresis is commonly applied for the analysis of pharmaceutical products due to its high separation efficiency and selectivity. For this purpose, electrospray-ionization-(ESI)-interfering additives or electrolytes are often required, which complicates the identification of impurities and degradation products by mass spectrometry (MS). Here, a capillary zone electrophoresis (CZE) method with ultraviolet (UV) absorption detection for the simultaneous determination and quantification of ascorbic acid and acetylsalicylic acid in effervescent tablets was developed. Related degradation products were identified via CZE-CZE-MS. Systematic optimization yielded 100 mM tricine (pH = 8.8) as appropriate background electrolyte, resulting in baseline separation of ascorbic acid, acetylsalicylic acid, and related anionic UV-active degradation products. The CZE-UV method was successfully validated regarding the guidelines of the Food and Drug Administration. The validated method was applied to trace the degradation rate of the active pharmaceutical ingredients at defined ambient conditions. A heart-cut CZE-CZE-MS approach, including a 4-port-nL-valve, was performed for the identification of the observed degradation products. This 2D setup enables a precise cutting of accurate sample volumes (20nL) and the independent operation of two physically separated CZE dimensions, which is especially beneficial regarding MS detection. Hence, the ESI-interfering tricine electrolyte components were separated from the analytes in a second electrophoretic dimension prior to ESI-MS detection. The degradation products were identified as salicylic acid and mono- and diacetylated ascorbic acid. This setup is expected to be generally applicable for the mass spectrometric characterization of CZE separated analytes in highly ESI-interfering electrolyte systems. Graphical Abstract A CZE-UV method for the quantification of effervescent tablet ingredients and degradation products was developed and validated. In order to identify unknown degradation products separated in the CZE-UV, a 2D heart-cut approach was performed applying a mechanical 4-port-valve. The unknown substances were transferred from the 1st to the 2nd dimension followed by the separation of ESI-interfering tricine from the analytes prior to mass spectrometric detection.

Targeted multidimensional gas chromatography using a heart-cutting device and cryogenic focusing for the determination of benzophenone derivatives in foodstuffs

Photoinitiators are used to promote the polymerization process during the curing of varnishes or inks on cartonboard packaging. Depending on storage conditions and shelf life, these substances are able to migrate through the packaging layer into the foodstuff. This type of contamination phenomenon is therefore becoming a critical issue in terms of food safety. In order to tackle this problem, a fast and selective method was developed for the determination of benzophenone and three methylbenzophenone isomers in cereal-based foodstuffs and their cardboard packaging. Food samples or packages were efficiently extracted by pressurized liquid extraction using acetonitrile, and the extracts were directly injected onto the analytical system. The analysis was performed by multidimensional gas chromatography-mass spectrometry using a heart-cutting approach to reduce the background noise from complex matrices. The strategy employed two distinct cuts each containing its proper deuterated internal standard leading to accurate quantification. By integrating a cryofocusing effect, an enhancement in signal/noise ratio was achieved by a factor >10, which markedly decreased the sensitivity threshold. Moreover, baseline separation of the critical isomers allowed their unequivocal determination. The method was fully validated on cereal-based foodstuffs based upon an analysis of variance, and excellent performances were obtained at the decision limit making this method well suited for official food controls.

Application to SFC-GC × GC to heavy petroleum fractions analysis

The new multidimensional chromatographic instrument SFC-GC×GC enables the temporary storage of SFC fractions inside sampling loops for subsequent analyses by GC×GC. The interface was configured for the characterization of heavy petroleum fractions. The SFC dimension was adapted to perform a group type separation into saturated, unsaturated and polar fractions. The GC×GC dimension was set in order to realize high temperature separations with two sets of columns. In a heart-cutting approach, an extended quantitative characterization of hydrocarbons contained in vacuum gas oil (VGO) was performed in a single run. Subsequently, in a comprehensive approach, quantitative 3D contour plots were built for the same VGO sample. The described application of the SFC-×2GC×GC instrument represents an innovative, rapid and automatic way for obtaining extended quantitative characterizations of heavy petroleum fractions.

Two‐dimensional HPLC analysis of oligostyrenes: Comprehensive and online heart‐cutting techniques

2-D HPLC incorporating two reversed phase (RP) environments was employed for the isolation of oligomers and their diastereomers of low molecular weight oligostyrenes. The operation of a comprehensive method of analysis was compared to a heart-cutting approach. The comprehensive approach employed a high resolution diastereomer separation in the first dimension and a low peak capacity C18, high speed separation according to molecular weight. Because of solvent incompatibility between the dimensions in the comprehensive method, successful separation of the diastereomers of the oligomers was not possible. The heart-cutting approach used a C18 monolith in the first dimension, which was selective only for molecular weight. Entire molecular weight fractions were then transported to the second dimension in an online heart-cutting process for the separation of diastereomers. The heart-cutting process was more successful in that 228 components of the 511 within the sample were recognized. This series of separations was undertaken in less than 6 h.

Determination of characteristic odorants from Harmonia axyridis beetles using in vivo solid-phase microextraction and multidimensional gas chromatography–mass spectrometry–olfactometry

Homeowners, small fruit growers, and wine makers are concerned with noxious compounds released by multicolored Asian ladybird beetles ( Harmonia axyridis, Coleoptera: Coccinellidae). A new method based on headspace solid-phase microextraction (HS-SPME) coupled with multidimensional gas chromatography–mass spectrometry–olfactometry (MDGC–MS–O) system was developed for extraction, isolation and simultaneous identification of compounds responsible for the characteristic odor of live H. axyridis. Four methoxypyrazines (MPs) were identified in headspace volatiles of live H. axyridis as those responsible for the characteristic odor: 2,5-dimethyl-3-methoxypyrazine (DMMP), 2-isopropyl-3-methoxypyrazine (IPMP), 2- sec-butyl-3-methoxypyrazine (SBMP), and 2-isobutyl-3-methoxypyrazine (IBMP). To the best of our knowledge this is the first report of H. axyridis releasing DMMP and the first report of this compound being a component of the H. axyridis characteristic odor. Besides the MPs, 34 additional compounds were also identified. Quantification of three MPs (IPMP, SBMP and IBMP) emitted from live H. axyridis were performed using external calibration with HS-SPME and direct injections. A linear relationship ( R 2 > 0.9951 for all 3 MPs) between MS response and concentration of a standard was observed over a concentration range from 0.1 ng L −1 to 0.05 μg L −1 for HS-SPME–GC–MS. The method detection limits (MDL) based on multidimensional GC–MS with narrow heart-cut approach for three MPs were estimated to be between 0.020 and 0.022 ng L −1. This represents a 38.9–52.4% improvement in sensitivity compared to GC–MS with full heart-cut method. This methodology is applicable for in vivo determination of odor-causing chemicals associated with emissions of volatiles from insects.

Heart-cutting two-dimensional electrophoresis in a single capillary

The possibility of performing two-dimensional capillary electrophoresis (2D-CE) separations in a single capillary was investigated. For this purpose, a fraction stemming from the first dimension of the separation was selected and isolated in the capillary by evacuating out of the capillary the other undesirable compounds. Next, the isolated fraction was submitted to a second separation medium that was introduced in the capillary by electroosmotic flow. The second separation medium was able to reach the isolated fraction since the solutes were migrating in counter-electroosmotic mode. Since only one fraction is submitted to the second dimension of the separation, this new methodology is closer to a heart-cutting approach than to a true comprehensive 2D separation. However, it has the advantage of not requiring any special coupling device between capillaries since the two dimensions of the separation are performed in the same capillary. In this work, a simple mixture of synthetic polymers taken as model compounds was separated according to: (i) the charge density by free solution CE in the first dimension, and (ii) the molar masses by CE in the presence of an entangled polymer solution in the second dimension. The different strategies that were investigated to isolate the fraction at the end of the first dimension are described in detail. The influence of important experimental parameters (capillary diameter, applied pressure for mobilizing the solutes, diffusion coefficients of the solutes) on the performances of the two-dimensional separations were studied. A careful attention was paid to the influence of these parameters on the efficiency of the separations. The experimental results demonstrate that heart-cutting 2D electrophoretic separations can be performed in a capillary format using a single capillary.

Separation of oligostyrene isomers in a complex mixture using two-dimensional heart-cutting reversed-phased liquid chromatography

The development of a two-dimensional liquid chromatographic system requires a process of assessment that can yield an optimum performing system with minimal experimental evaluation. Information Theory and a geometric approach to Factor Analysis are two techniques that when used in combination, provide important information on the expected two-dimensional performance. In the present study, we compare the predicted separation performance of two-dimensional systems that have been subjected to analysis by Information Theory and Factor Analysis to that of actual chromatographic separation performance. Our test separation comprised a mixture of 32 oligostyrene structural isomers and stereoisomers. The optimal combination as determined by Information Theory and Factor Analysis consisted of a C18 column with a methanol mobile phase in the first dimension and a carbon clad zirconia column with an acetonitrile mobile phase in the second dimension. This system was also shown to be the most successful practical system when a heart-cutting approach was employed. The practical results were in total agreement with the results from Information Theory and Factor Analysis. The number of isomers resolved using this system was 27. A second system, namely one comprising of a C18 column and methanol mobile phase in the first dimension and a carbon clad zirconia column with a methanol mobile phase in the second dimension was also predicted to be a system with high separation potential. However, practical assessment of this system did not realise the theoretical predictions, largely due to the long separation times required in the second dimension. Furthermore, all combinations that employed a C18 column with an acetonitrile mobile phase in the first dimension failed to realise the theoretical separation potential due to high solute crowding, low orthogonality and a disordered arrangement of bands along the first separation axis. This was also predicted by the theoretical assessment.

Development of a LC–LC–MS complete heart-cut approach for the characterization of pharmaceutical compounds using standard instrumentation

A total heart-cut LC–LC–MS technique is described for the analysis of pharmaceutical compounds. Utilizing ordinary LC–MS equipment, a standardized multi-dimensional, highly selective approach was developed. The system was developed on synthetic mixtures containing ten active pharmaceutical compounds. The methodology would be further deployed for the impurity characterization of batches of active chemical, process intermediates, and reference standards. Column-switching was employed to heart-cut sequential fractions from one set of LC conditions to another set of LC conditions. Two high efficiency columns, including the Merck Chromolith® C18, for fast LC–LC analysis were used. The system provides a highly automated, turnkey, multi-dimensional approach applicable to a variety of complex mixtures with little user intervention. It is envisioned that this idea could be viewed as a platform for other types of LC–LC analyses including peptide and achiral–chiral analyses.