On-line Chromatographic Separation Research Articles

Determination of Lipophilic Marine Biotoxins in Shellfish by Online Turbulent Flow Chromatography Coupled to Liquid Chromatography–Tandem Mass Spectrometry

A novel method based on online turbulent flow chromatography (TFC) combined with liquid chromatography–tandem mass spectrometry (LC–MS/MS) has been established for simultaneous quantitative determination of 11 lipophilic marine toxins (OA, DTX1, DTX2, YTX, hYTX, AZA1, AZA2, AZA3, PTX2, SPX1, and GYM) in shellfish. Sample preparation, including extraction with 75% methanol aqueous solution, centrifugation, and filtration, was followed by online cleanup using TFC. The total run time was within 15.5 min, including online purification, chromatographic separation, and re-equilibration of the TFC system. Various experimental parameters including extraction, purification, separation, and detection were optimized separately in this study. The developed method was validated, and good performance characteristics were obtained. The linear regression coefficients (R2) of matrix-match calibration standard curves established for quantification were higher than 0.9945. The limits of detection and quantification of this method ranged between 0.5 and 4.0, and 1.5 and 10.0 µg/kg, respectively. The average recoveries of spiked target compounds with different concentrations ranged from 80.1 to 113.8%, the intra- and inter-day precision were lower than 12.7% and 14.9%, respectively. The matrix effects were in the range of 76.1–115.9%. The method accuracy was also confirmed with certified reference materials. The results indicate that the developed method has great potential for the routine laboratory analysis or emergency detection of lipophilic marine toxins in shellfish.

Position-specific 13 C/12 C analysis of amino acid carboxyl groups - automated flow-injection analysis based on reaction with ninhydrin.

RationaleThe fundamental level of stable isotopic knowledge lies at specific atomic positions within molecules but existing methods of analysis require lengthy off‐line preparation to reveal this information. An automated position‐specific isotope analysis (PSIA) method is presented to determine the stable carbon isotopic compositions of the carboxyl groups of amino acids (δ 13CCARBOXYL values). This automation makes PSIA measurements easier and routine.MethodsAn existing high‐performance liquid chromatography (HPLC) gas handling interface/stable isotope ratio mass spectrometry system was modified by the addition of a post‐column derivatisation unit between the HPLC system and the interface. The post‐column reaction was optimised to yield CO2 from the carboxyl groups of amino acids by reaction with ninhydrin.ResultsThe methodology described produced δ 13CCARBOXYL values with typical standard deviations below ±0.1 ‰ and consistent differences (Δ 13CCARBOXYL values) between amino acids over a 1‐year period. First estimates are presented for the δ 13CCARBOXYL values of a number of internationally available amino acid reference materials.ConclusionsThe PSIA methodology described provides a further dimension to the stable isotopic characterisation of amino acids at a more detailed level than the bulk or averaged whole‐molecule level. When combined with on‐line chromatographic separation or off‐line fraction collection of protein hydrolysates the technique will offer an automated and routine way to study position‐specific carboxyl carbon isotope information for amino acids, enabling more refined isotopic studies of carbon uptake and metabolism.

Monolithic molecularly imprinted polymeric capillary columns for isolation of aflatoxins

Monolithic molecularly imprinted polymers extraction columns have been prepared in fused-silica capillaries by UV or thermal polymerization in a two-step process. First, a poly-(trimethylolpropane trimethacrylate) (polyTRIM) core monolith was synthesized either by UV or thermal polymerization. Then it was grafted with the mixture of methacrylic acid (MAA) as a functional monomer, ethylene dimethacrylate (EDMA) as a cross-linking agent, 5,7-dimethoxycoumarin (DMC) as an aflatoxin-mimicking template, toluene as a porogen solvent and 2,2-azobis-(2-methylpropionitrile) (AIBN) as an initiator of the polymerization reaction. Different thermal condition of the photografting and different concentrations of the grafting mixture were tested during polymerization. The extraction capillary columns were evaluated in the terms of their hydrodynamic and chromatographic properties. Retention coefficients for aflatoxin B1 and DMC were used for assessment of the selectivity and imprinting factor. The obtained results indicate that the temperature of photografting and concentration of the grafting mixture are key parameters that determine the quality of the prepared MIPs. From the MIP columns characterized by the highest permeability the column of the highest imprinting factor was applied for isolation of aflatoxins B1, B2, G1 and G2 from the model aqueous sample followed by on-line chromatographic separation. The process was performed using a micro-MISPE-microLC-LIF system of a novel design, which allowed for detection of the eluates from the sample preparation part as well as from the chromatographic separation.

Detailed Glycan Structural Characterization by Electronic Excitation Dissociation

The structural complexity and diversity of glycans parallel their multilateral functions in living systems. To better understand the vital roles glycans play in biological processes, it is imperative to develop analytical tools that can provide detailed glycan structural information. This was conventionally achieved by multistage tandem mass spectrometry (MS(n)) analysis using collision-induced dissociation (CID) as the fragmentation method. However, the MS(n) approach lacks the sensitivity and throughput needed to analyze complex glycan mixtures from biological sources, often available in limited quantities. We define herein the critical parameters for a recently developed fragmentation technique, electronic excitation dissociation (EED), which can yield rich structurally informative fragment ions during liquid chromatographic (LC)-MS/MS analysis of glycans. We further demonstrate that permethylation, reducing end labeling and judicious selection of the metal charge carrier, can greatly facilitate spectral interpretation. With its high sensitivity, throughput, and compatibility with online chromatographic separation techniques, EED appears to hold great promise for large-scale glycomics studies.

Direct Characterization of Bulk Samples by Internal Extractive Electrospray Ionization Mass Spectrometry

A straight-forward analytical strategy called internal extractive electrospray ionization mass spectrometry (iEESI-MS), which combines solvent extraction of chemicals inside a bulk sample with in situ electrospray ionization mass spectrometry, has been established to directly characterize the interior of a bulk sample with molecular specificity. The method allows both qualitative and quantitative analysis of analytes distributed in a 3-dimensional volume (e.g., 1 ~ 100 mm3) of various synthetic and biological matrices (e.g., chewing gum, leaves, fruits, roots, pork, lung tissues) without either mashing the sample or matrix separation. Using different extraction solvents, online chromatographic separation of chemicals inside the sample volume was observed during iEESI-MS analysis. The presented method is featured by the high speed of analysis, high sensitivity, low sample consumption and minimal sample preparation and/or degradation, offering unique possibilities for advanced applications in plant science, clinical diagnosis, catalyst studies, and materials science.

Comprehensive Characterization of Posttranslational Modifications of Histones Using a Novel High‐Throughput Middle‐Down Proteomics Approach

Until recently, histones have mostly been characterized by either bottom‐up or top‐down mass spectrometric analysis. While the bottom‐up approach is more sensitive, the important connectivity of the posttranslational modifications of the N‐terminus is often lost. This connectivity is maintained in top‐down proteomics, but the sensitivity of this approach is lacking. Middle‐down proteomics is a compromise between bottom‐up and top‐down analysis using a limited digestion that creates peptides large enough to maintain the important connectivity in the N‐terminal tail of histones. To achieve a high‐throughput method, middle‐down proteomics was coupled with on‐line chromatographic separation using electron capture dissociation (ECD), a fragmentation technique that preserves the posttranslational modifications. Taking advantage of the high mass accuracy in the FTICR mass analyzer, several histone variants were identified and modification sites were successfully localized, including single, multiple and positional isomeric PTM sites. In general, protein sequence coverage was well over 50%, reaching 100% in many instances.Middle‐down nano‐LC‐ECD MS/MS approach enables the comprehensive, high‐throughput analysis of unfractionated histones and reveals global modification profiles and localization of PTMs.

Data-Dependent Middle-Down Nano-Liquid Chromatography–Electron Capture Dissociation-Tandem Mass Spectrometry: An Application for the Analysis of Unfractionated Histones

Middle-down mass spectrometry (MS) combined with electron capture dissociation (ECD) represents an attractive method for characterization of proteins and their post-translational modifications (PTMs). Coupling online chromatographic separation with tandem mass spectrometry enables a high-throughput analysis, while improving sensitivity of the electrosprayed peptides and reducing sample amount requirements. However, middle-down ECD has not been thus far coupled with online chromatographic separation. In this work, we examine the feasibility of coupling middle-down ECD with online nanoflow-liqiud chromatography (nano-LC) for the analysis of large, >3 kDa, and highly modified polypeptides in a data-dependent acquisition mode. We evaluate the effectiveness of the method by analyzing peptides derived from Asp-N and Glu-C digestions of unfractionated histones from calf thymus and acid-extracted histones from HeLa, MCF-7, and Jurkat cells. Our results demonstrate that middle-down ECD is compatible with online chromatographic separation, providing high peptide and protein sequence coverage while allowing precise mapping of PTM sites. The high mass accuracy, obtained by the ICR mass analyzer, for both the precursor and product ions greatly increases confidence in peptide identification, particularly for modified peptides. Overall, for all samples examined, several histone variants were identified and modification sites were successfully localized, including single, multiple, and positional isomeric PTM sites. The vast majority of the identified peptides were in the mass range from 3 to 9 kDa. The data presented here highlight the feasibility and utility of nano-LC-ECD-MS/MS for high-throughput middle-down analysis.

A Simulated MS/MS Library for Spectrum-to-spectrum Searching in Large Scale Identification of Proteins

Identifying peptides from mass spectrometric fragmentation data (MS/MS spectra) using search strategies that map protein sequences to spectra is computationally expensive. An alternative strategy uses direct spectrum-to-spectrum matching against a reference library of previously observed MS/MS that has the advantage of evaluating matches using fragment ion intensities and other ion types than the simple set normally used. However, this approach is limited by the small sizes of the available peptide MS/MS libraries and the inability to evaluate the rate of false assignments. In this study, we observed good performance of simulated spectra generated by the kinetic model implemented in MassAnalyzer (Zhang, Z. (2004) Prediction of low-energy collision-induced dissociation spectra of peptides. Anal. Chem. 76, 3908-3922; Zhang, Z. (2005) Prediction of low-energy collision-induced dissociation spectra of peptides with three or more charges. Anal. Chem. 77, 6364-6373) as a substitute for the reference libraries used by the spectrum-to-spectrum search programs X!Hunter and BiblioSpec and similar results in comparison with the spectrum-to-sequence program Mascot. We also demonstrate the use of simulated spectra for searching against decoy sequences to estimate false discovery rates. Although we found lower score discrimination with spectrum-to-spectrum searches than with Mascot, particularly for higher charge forms, comparable peptide assignments with low false discovery rate were achieved by examining consensus between X!Hunter and Mascot, filtering results by mass accuracy, and ignoring score thresholds. Protein identification results are comparable to those achieved when evaluating consensus between Sequest and Mascot. Run times with large scale data sets using X!Hunter with the simulated spectral library are 7 times faster than Mascot and 80 times faster than Sequest with the human International Protein Index (IPI) database. We conclude that simulated spectral libraries greatly expand the search space available for spectrum-to-spectrum searching while enabling principled analyses and that the approach can be used in consensus strategies for large scale studies while reducing search times.

Implementation of Electron-Transfer Dissociation on a Hybrid Linear Ion Trap−Orbitrap Mass Spectrometer

We describe the adaptation of a hybrid quadrupole linear ion trap-orbitrap mass spectrometer to accommodate electron-transfer ion/ion reactions (ETD) for peptide and protein characterization. The method utilizes pulsed, dual electrospray ion sources and requires minimal instrument modification. Switching between cation and reagent anion injection schemes is automated and accomplished within a few hundred milliseconds. Ion/ion reactions are conducted within the linear ion trap, after which the c- and z-type product ions are passed to the orbitrap for high-resolution m/z analysis. With this arrangement, mass accuracies are typically measured to within 2 ppm at a resolving power of approximately 60 000. Using large peptides and intact proteins, we demonstrate such capabilities will accelerate our ability to interrogate high-mass species. To illustrate compatibility with automated data-dependent analysis and subsequent data processing, we couple the technique with an online chromatographic separation of a yeast whole-cell lysate followed by peptide identification using ProSight PC. Fairly long pulsing times and relatively low ET efficiency, as compared to conventional ETD instrumentation, are the main drawbacks of this approach. Still, our results suggest that the implementation of ETD on sensitive, high-resolution, and high-mass accuracy hybrid instrumentation, such as the orbitrap, will substantially propel the emergent fields of middle- and top-down proteomics.

An overview of sequential injection chromatography

New generation of sequential injection analysis (SIA) called sequential injection chromatography (SIC) has already been consolidated as a good alternative of high performance liquid chromatography (HPLC) for fast analysis of simple samples. Benefits of flow methods are automation, miniaturization and low sample and mobile phase consumption. Implementation of short monolithic chromatographic column into SIA opens new area—on-line chromatographic separation of multi-compound sample in low-pressure flow system, with the advantage of flow programming and possibility of sample manipulation. In the presented review the potential of SIC and its comparison with HPLC for determination of pharmaceutical mixtures is discussed and outlines past and recent trends focused on separation with SIC.

Membrane in tandem with a helical sorbent trap as continuous sampling technique of the polyvinyl chloride thermo-oxidative degradation products for their on-line gas chromatographic monitoring

A flat membrane in tandem with a helical sorbent trap has been used for continuous sampling of the volatile organic products generated in the thermal degradation process of the polyvinyl chloride (PVC) in air, followed by on-line gas chromatographic separation and mass spectrometric identification. The membrane and trap tandem makes automatic collection, concentration, and injection of PVC volatile and semivolatile degradation products, and it is simple in terms of instrumentation and operation. The poly(dimethylsiloxane) (PDMS) membrane used in this study shows a low permeation for oxygenated derivatives and a high permeation for volatile aromatic and non-aromatic hydrocarbon, and chlorinated hydrocarbons. Consequently, the final chromatogram is significantly simplified. By heating the trap at fixed intervals of time, consecutive gas chromatograms are obtained in the monitoring process. The sensitivity of the method depends on the parameters that affect the time of trapping, and the permeation through the membrane.

Quantitative determination of saturated oligogalacturonic acids in enzymatic digests of polygalacturonic acid, pectin and carrot pomace by on-line LC-ESI-MS.

A method for the quantitative determination of oligogalacturonic acids (OGAs) by on-line high-performance liquid chromatographic (HPLC) separation and mass spectrometric detection via an electrospray interface (ESI-MS) without additional desalting steps was developed. Saturated OGAs up to a degree of polymerization (dp) of 3 were quantified by comparison with reference compounds. The calibration plots showed high linearity (R(2)>0.99), and the detection limits for dp 1, 2, and 3 were 11, 28, and 6 ng per injection, respectively. Non- and partially methyl-esterified OGAs with a dp of 3 and 4 were calculated semi-quantitatively as dp 3. The analytical system was used for the quantification of OGAs of digests obtained by incubation of polygalacturonic acid, pectin, and carrot pomace with commercial enzyme preparations. Furthermore, methyl-esterified OGAs up to a dp of 12 containing up to 4 methyl esters were detected in a pectin digest.

Development of a liquid chromatography-electrospray-tandem mass spectrometry method for the quantitative determination of benzoxazinone derivatives in plants.

A new method for the quantification of benzoxazinone derivatives in extracts of wheat foliage and root samples using liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS-MS) is described. Using this method, the characterization, separation, and quantitative detection of a mixture of six naturally occurring 1,4-benzoxazin-3(4H)-one derivatives, including the hydroxamic acids (DIMBOA, DIBOA), lactams (HBOA, and HMBOA), benzoxazilinones (BOA, MBOA), and two synthetic methoxylated variations of DIBOA and HBOA, was achieved. The application of a novel, highly modified reversed-phase LC column, the dodecyl (C12) TMS end-capped Synergi MAX-RP, enhanced the on-line chromatographic separation through improvements to component resolution, analyte stability and peak shape and also to the column lifetime. The complete ESI-MS-MS precursor-product ion fragmentation pathways for the benzoxazinone derivatives are described for the first time and used to deduce a generic fragmentation pattern for the compound class. Characteristic transitions for the benzoxazinones were thus used in the developed analytical method enabling reliable quantification with simultaneous screening for other potentially present derivatives, while eliminating interferences from other coeluting contaminants from the complex plant extract matrix. Quantitative analysis was done in the multiple reaction monitoring mode, using two specific combinations of a precursor-product ion transitions for each compound. The ESI-MS-MS detection method offered improvements to the sensitivity and selectivity, as compared with previously applied LC methods, with detection limits down to 0.002-0.023 ng/microL. The developed method was demonstrated by analyzing foliages and roots of six different wheat cultivars using pressurized liquid extraction-solid-phase extraction cleanup-LC-ESI-MS-MS. The analytes were detected in the range of 0.7-207 microg/g of dry weight.

Automated quantitative determination of the new renin inhibitor CGP 60536 by high-performance liquid chromatography

A fully automated high-performance liquid chromatography method with fluorescence detection for the determination of the renin inhibitor CGP 60536 in animal and human plasma and urine has been developed and validated. After addition of an internal standard, the compounds were automatically extracted from 400 μl of plasma or urine with methyl alcohol–acetic acid (99:1, v/v) on 100-mg Bond-Elut CN cartridges using the Gilson ASPEC system. The on-line chromatographic separation was performed on a LiChrospher 100 RP8 5-μm particle size packed analytical column (25×0.4 cm I.D.). The mobile phase consisted of acetonitrile–0.01 M potassium dihydrogenphosphate (65:35, v/v) at a flow-rate of 0.8 ml/min. The analytes were detected using a fluorescence detector at excitation and emission wavelengths of 280 and 330 nm, respectively. The limit of quantitation was established at 4.5 ng/ml in plasma (accuracy 106% and precision 1%), and 9.0 ng/ml in urine (accuracy 101% and precision 13%). The method was applied to the investigation of the pharmacokinetics of CGP 60536.

External Accumulation of Ions for Enhanced Electrospray Ionization Fourier Transform Ion Cyclotron Resonance Mass Spectrometry

Electrospray ionization (ESI) in combination with Fourier transform ion cyclotron resonance (FTICR) mass spectrometry provides for mass analysis of biological molecules with unrivaled mass accuracy, resolving power and sensitivity. However, ESI FTICR MS performance with on-line separation techniques such as liquid chromatography (LC) and capillary electrophoresis has to date been limited primarily by pulsed gas assisted accumulation and the incompatibility of the associated pump-down time with the frequent ion beam sampling requirement of on-line chromatographic separation. Here we describe numerous analytical advantages that accrue by trapping ions at high pressure in the first rf-only octupole of a dual octupole ion injection system before ion transfer to the ion trap in the center of the magnet for high performance mass analysis at low pressure. The new configuration improves the duty cycle for analysis of continuously generated ions, and is thus ideally suited for on-line chromatographic applications. LC/ESI FTICR MS is demonstrated on a mixture of 500 fmol of each of three peptides. Additional improvements include a fivefold increase in signal-to-noise ratio and resolving power compared to prior methods on our instrument.

On-line chromatographic separation and determination of chromium(III) and chromium(VI) with preconcentration of the chromium(III) using potassium hydrogen phthalate, in various samples by flame atomic absorption spectrometry

A method has been developed for the on-line LC separation of CrIII on a reversed-phase C18 column in the presence of potassium hydrogen phthalate, which is a recently introduced reagent for chromatography. It is much more selective for CrIII in contrast to many other complex-forming agents discussed in the literature, so that in its presence virtually no ions are sorbed onto the C18 column except for CrIII. Hence almost no interferences due to the matrix are found during the detection of CrIII. The CrIII sorbed onto the column was eluted into the flame of an atomic absorption spectrometer using a mixture of methanol and water [80–20(%(v/v))]. With 100 µl of sample solution the detection limits (3σ) were found to be 0.024 and 0.075 mg l–1 for the separation of CrIII and CrVI with FAAS detection and the period needed for separation was under 60 s. For the preconcentration of the CrIII content of a 5 ml sample solution, the detection limit (3σ) was 0.73 ng ml–1, the accuracy of the measurement was 3.8% RSD for the preconcentration of 20 ng ml–1 of CrIII. The sensitivity of the method enabled the determination of the CrIII and CrVI content of surface waters and various samples of natural origin at concentrations below ng ml–1 to be made. After reduction of CrVI to CrIII with ascorbic acid and its preconcentration, the total Cr content of various samples could be determined.

Determination of the anti-platelet-activating factor BN-50727 and its metabolites in human plasma by high-performance liquid chromatography-solid-phase extraction.

A sensitive and selective HPLC-solid-phase extraction procedure was developed for the determination of platelet-activating factor antagonist BN-50727 and its metabolites in human plasma. The procedure consisted of an automated solid-phase extraction of the drug and metabolites on disposable propylcarboxylic acid cartridges, followed by on-line chromatographic separation. The method was linear from 3.75 to 2400 ng/ml and the limit of quantitation for BN-50727 in plasma samples was 3.75 ng/ml. The within-run precision of the method, expressed as relative standard deviation, ranged from 2.1 to 8.1%. The accuracy, expressed as relative error, ranged from -3.5 to 4.0%. For the main metabolite, the O-demethylated BN-50727 product, the method was linear from 7.5 to 2400 ng/ml and the limit of quantitation in plasma was 7.5 ng/ml. The within-run precision ranged from 2.1 to 11.0% and the accuracy from -5.3 to 1.1%. This paper describes the validation of the analytical methodology for the determination of BN-50727 in human plasma and also of its metabolites. The method has been used to follow the time course of BN-50727 and its metabolites in human plasma after administration of single and multiple doses.

Isolation and Monitoring of the Endohedral Metallofullerenes Y@C82 and Sc3@C82: Online Chromatographic Separation with EPR Detection

ADVERTISEMENT RETURN TO ISSUEPREVArticleNEXTIsolation and Monitoring of the Endohedral Metallofullerenes [email protected] and [email protected]: Online Chromatographic Separation with EPR DetectionS. Stevenson, H. C. Dorn, P. Burbank, K. Harich, Z. Sun, C. H. Kiang, J. R. Salem, M. S. DeVries, P. H. M. van Loosdrecht, and . et al.Cite this: Anal. Chem. 1994, 66, 17, 2680–2685Publication Date (Print):September 1, 1994Publication History Published online1 May 2002Published inissue 1 September 1994https://doi.org/10.1021/ac00089a014RIGHTS & PERMISSIONSArticle Views162Altmetric-Citations39LEARN ABOUT THESE METRICSArticle Views are the COUNTER-compliant sum of full text article downloads since November 2008 (both PDF and HTML) across all institutions and individuals. These metrics are regularly updated to reflect usage leading up to the last few days.Citations are the number of other articles citing this article, calculated by Crossref and updated daily. Find more information about Crossref citation counts.The Altmetric Attention Score is a quantitative measure of the attention that a research article has received online. Clicking on the donut icon will load a page at altmetric.com with additional details about the score and the social media presence for the given article. Find more information on the Altmetric Attention Score and how the score is calculated. Share Add toView InAdd Full Text with ReferenceAdd Description ExportRISCitationCitation and abstractCitation and referencesMore Options Share onFacebookTwitterWechatLinked InReddit PDF (557 KB) Get e-Alerts Get e-Alerts

Simultaneous determination of methyl-, ethyl-, phenyl- and inorganic mercury by cold vapour atomic absorption spectrometry with on-line chromatographic separation

A fully automated system for on-line high-performance liquid chromatographic separation, reduction and determination by cold vapour atomic absorption spectrometry (CVAAS) of methyl-, ethyl-, phenyl- and inorganic mercury is described. Reversed-phase chromatography on an ODS column and elution with an acetonitrile-water-ammonium tetramethylenedithiocarbamate buffered mixture was investigated with or without ammonium tetramethylenedithiocarbamate precomplexation. A simple glass flow cell, properly designed as an interface between the chromatographic system and CVAAS, is proposed. The influence of the composition of the eluent, reducing solution and stripping gas flow-rate was investigated and optimized in order to obtain better detection limits. The method, applied to synthetic mixtures and natural samples of tap water furnished satisfactory results. It is shown that, with the on-line preconcentration procedure, sub-ng/ml concentration levels can be determined for all the species considered.

Preliminary study of metals in proteins by high-performance liquid chromatography-inductively coupled plasma mass spectrometry using multi-element time-resolved analysis.

The feasibility of multi-element and multi-isotope determinations during the on-line chromatographic separation of protein matrices was investigated using time-resolved software with inductively coupled plasma mass spectrometry. Size-exclusion chromatography was used to separate a mixture of known proteins and the behaviour of associated elements was monitored. It was possible to scan either the entire mass range or a selected number of isotopes. Reversed-phase chromatography was used to separate Zn-containing species in chicken meat that had been intrinsically and extrinsically labelled with stable isotopes of Zn. The ratio obtained for the intrinsic label agreed with that obtained by other workers using thermal ionization mass spectrometry. Time-resolved analysis permitted rapid data acquisition, thus reducing the number of chromatographic runs required. However, at present, data handling can be time consuming. Further software developments will add to the advantages that this technique offers.