Splitless on-line coupling of capillary high-performance liquid chromatography,capillary electrochromatography and pressurized capillary electrochromatography with nuclear magnetic resonance spectroscopy

A mixture of unsaturated fatty acid methyl esters was separated with a new splitless capillary set-up. With the employed apparatus configuration different capillary separation techniques such as capillary high-performance liquid chromatography (cHPLC), capillary electrochromatography (CEC) and pressurized capillary electrochromatography (pCEC) could be applied. The detection and identification of the sample compounds were accomplished by hyphenating these capillary separation techniques with nuclear magnetic resonance (NMR) spectroscopy using a novel configuration of the detection capillary set-up. Using modified electrokinetically driven separation techniques, the electric field was applied solely across the separation column. With this improved interface for capillary liquid chromatography-NMR on-line coupling, the stereochemical assignment of the cis and trans configuration of unsaturated fatty acids could be easily accomplished. Finally, the results of cHPLC-NMR, CEC-NMR and pCEC-NMR coupling experiments were compared.Dedicated to Professor Günter Häfelinger on the occasion of his 65th birthday     

Comprehensive two-dimensional liquid chromatography coupled with mass spectrometry

In this review, instrumental aspects of comprehensive two-dimensional liquid chromatography coupled with mass spectrometry are presented. The milestones of LC×LC are briefly summarized. Instrument configuration, selection of experimental conditions, the different interfaces used in the system and the current applications of LC×LC–MS systems are described.     

Fingerprint analysis of Rhizoma chuanxiong by pressurized capillary electrochromatography and high-performance liquid chromatography

Pressurized capillary electrochromatography (pCEC) and high-performance liquid chromatography (HPLC) were used simultaneously to establish fingerprints of Rhizoma chuanxiong. Ten batches of Rhizoma chuanxiong collected from different regions in China were used to obtain the characteristic pCEC and HPLC fingerprints using a standardized procedure of sample preparation and analysis. A total of 22 common peaks were isolated within 60 min by pCEC and 16 common peaks by HPLC within 65 min. The fingerprints of Rhizoma chuanxiong were then used to identify the raw herbs from different sources in China. The two proposed methods demonstrated good stability and reproducibility with RSD less than 5% for retention time in pCEC and in HPLC, respectively. Finally, the data from the analyses of 10 batches of Rhizoma chuanxiong by pCEC and HPLC were all processed with similarity analysis with two mathematical methods, correlation coefficient and the included angle cosine. The fingerprints of Rhizoma chuanxiong established with pCEC and HPLC are suitable to identify samples from different sources and can be used to control the quality of raw herbs.     

Strategy for metabonomics research based on high-performance liquid chromatography and liquid chromatography coupled with tandem mass spectrometry

Metabonomics, the study of metabolites and their roles in various disease states, is a novel methodology arising from the post-genomics era. This methodology has been applied in many fields. Current metabonomic practice has relied on mass spectrometry (MS), gas chromatography-mass spectrometry (GC-MS), and nuclear magnetic resonance (NMR) to analyze metabolites. In this study, a strategy was developed for applying high-performance liquid chromatography (HPLC) and LC-MS-MS to metabonomics research. One of the key problems to be solved in this strategy is to match the peaks between the chromatograms. A peak alignment algorithm has been developed to match the chromatograms before the pattern recognition. As an application example, the strategy described above was applied to metabonomics research on liver diseases, and the false-positive result of live cancer diagnosis from the hepatocirrhosis and hepatitis diseases was effectively reduced to 7.40%. Based on the pattern recognition, several potential biomarkers were found and further identified by the following LC-MS-MS experiments. The structures of eight potential biomarkers were given for distinguishing the liver cancer from the hepatocirrhosis and hepatitis diseases.     

Column liquid chromatography coupled on-line with mass spectrometry using post-column phosphate suppression

The coupling of column liquid chromatography on-line with mass spectrometry using post-column phosphate suppression is described. A membrane suppressor is used to remove the non-volatile phosphate ions from the mobile phase after the chromatographic separation prior to introduction into the mass spectrometer. Using post-column phosphate suppression by a membrane device phosphate concentrations up to 10 mM phosphate, at a mobile phase flow rate of 0.1 ml/min, could be removed for more than 99% without any decay of the sensitivity of the MS detection. Using a new antidepressant tetracyclic basic compound, ORG 4428, and some structure related compounds as model compounds electron impact as well as chemical ionisation spectra of all compounds could be obtained. The set-up could be used for several weeks running, using a phosphate containing mobile phase, without any loss of performance.     

On-line concentration of proteins in pressurized capillary electrochromatography coupled with electrospray ionization-mass spectrometry

Pressurized capillary electrochromatography (pCEC) and electrospray ionization-mass spectrometry (ESI-MS) have been hyphenated for protein analysis. Taken cytochrome c, lysozyme, and insulin as samples, the limits of detection (LODs) for absolute concentrations are 10(-11) mol (signal-to-noise ratio S/N = 3) with relative standard deviations (RSDs) of retention time and peak area, respectively, of less than 1.7% and 4.8%. In order to improve the detection sensitivity, on-line concentration by field-enhanced sample-stacking effect and chromatographic zone-sharpening effect has been developed, and parameters affecting separation and detection, such as pH and electrolyte concentration in the mobile phase, separation voltage, as well as enrichment voltage and time, have been studied systematically. Under the optimized conditions, the LODs of the three proteins could be decreased up to 100-fold. In addition, the feasibility of such techniques has been further demonstrated by the analysis of modified insulins at a concentration of 20 microg/mL.     

Electrodialytic sample treatment coupled on-line with high-performance liquid chromatography

Summary An electrodialytic sample treatment method coupled on-line with high-performance liquid chromatography (EDIST-HPLC) is discussed in this paper. The performance of EDIST as a function of the donor-phase (sample solution) flow rate, the voltage applied over the electrodialysis block, and the time of dialysis has been studied using the basic drug ephedrine as a model compound. Enrichment of the analyte by a factor of 10–20 was possible. The determination of human plasma spiked with ephedrine is briefly discussed.     

Comparison of capillary electrochromatography with high-performance liquid chromatography for the analysis of pirimicarb and related compounds.

The applicability of capillary electrochromatography (CEC) to the analysis of pirimicarb and structurally related pyrimidines has been investigated. Methods were developed to improve the separation of closely related compounds. Resolution was achieved both by the use of running buffers containing a mixture of two organic modifiers to increase selectivity and reduce retention times. Solvent composition step gradients were used to separate compounds of widely differing retention factors. A comparison has been made between HPLC and CEC using identical separation parameters and the same stationary phase, from which two important conclusions are drawn. First, it has been shown that values of k' for the compounds analyzed were the same in both techniques. Secondly, although it is evident that CEC produces higher efficiencies than HPLC when running buffers with high organic solvent content are used, as the aqueous content of the running buffer is increased the efficiencies achieved in CEC and HPLC converge until they become equivalent. This is contrary to the theoretical model which predicts efficiencies are inherently higher using electrically rather than pressure driven flow. Disadvantages of the limited control of flow-rate in CEC in comparison with HPLC, are shown.     

Characterization of mixtures of biocidal oligoguanidines by capillary electrophoresis and high-performance liquid chromatography coupled to mass spectrometry

The polycondensation of guanidine hydrochloride and 2,2′-(ethylenedioxy)bis(ethylamine) leads to various types of oligomeric guanidines exhibiting a broad spectrum of biocidal activities. In the present work a reversed-phase high-performance liquid chromatographic method with a gradient consisting of aqueous 0.1% trifluoroacetic acid and acetonitrile as mobile phase has been developed to separate these oligoguanidines according to type and chain length. The combination with electrospray mass spectrometry allowed the identification of the various compounds. By this technique, some structures already suggested in the literature could be confirmed, and several additional oligoguanidines not yet reported could be identified. As a complementary technique, capillary zone electrophoresis was investigated. Best results were obtained with carrier electrolytes consisting of phosphoric acid in water/acetonitrile mixtures. Although the number of peaks that could be separated by the electrophoretic method was considerably lower than in case of the chromatographic method, capillary electrophoresis in combination with UV detection at 195 nm may still be a fast method suitable for quantitation of some of the major compounds and for monitoring the reaction rate during the polycondensation reaction.     

Dynamics of capillary electrochromatography. II. Comparison of column efficiency parameters in microscale high-performance liquid chromatography and capillary electrochromatography.

In capillary electrochromatography (CEC) the flow of the mobile phase is generated by electrosmotic means in high electric field. This work compares band spreading measured experimentally in several packed capillaries with electrosmotic flow (EOF) and viscous flow under otherwise identical conditions. The data were fitted to the simplified van Deemter equation for the theoretical plate height, H = A + B/u + Cu, in order to evaluate parameters A and C in each mode of flow in the different columns. The ratio of these two parameters obtained with the same column in microscale HPLC (mu-HPLC) and CEC was used to quantify the attenuation of their contribution to band spreading upon changing from viscous flow (in mu-HPLC) to electrosmotic flow (in CEC). The capillary columns used in this study were packed with stationary phases of different pore sizes as well as retentive properties and measurements were carried out under different mobile phase conditions to examine the effects of the retention factor and buffer concentration. In the CEC mode, the value of both column parameters A and C was invariably by a factor of two to four lower than in the mu-HPLC mode. This effect may be attributed to the peculiarities of the EOF flow profile in the interstitial space and to the generation of intraparticle EOF inside the porous particles of the column packing. Thus, band spreading due to flow maldistribution and mass transfer resistances is significantly lower when the mobile phase flow is driven by voltage as in CEC, rather than by pressure as in mu-HPLC.     

Fully automated micro- and nanoscale one- or two-dimensional high-performance liquid chromatography system for liquid chromatography-mass spectrometry compatible with non-volatile salts for ion exchange chromatography

A one- or two-dimensional high performance liquid chromatography system for electrospray ionization mass spectrometers has been developed that is optimized for ion exchange and reversed phase separations. A unique and simple valve configuration permits the use of a variety of non-volatile salts; ammonium sulfate was used in an example of strong cation exchange separations. The system was designed and evaluated for both micro- and nanoflow chromatography. The peptide detection limit was approximately 100 fmol for micro- and 20 fmol for nanoflow, demonstrating the concentration and mass sensitivity improvements expected with nanoelectrospray ionization. The 1D/2D-HPLC MS system is fully automated for routine peptide analyses, compatible with direct injection of proteolytic digests, and exhibits chromatographic reproducibility and sensitivity. Software permits operator selection of either a 1D or 2D configuration with corresponding system parameters as required for individual samples. The hardware elements and resulting performance are described in this paper.     

Comprehensive two-dimensional chromatography and capillary electrophoresis coupled with tandem time-of-flight mass spectrometry for high-speed proteome analysis

A comprehensive two-dimensional capillary liquid chromatography and capillary zone electrophoresis system coupled with tandem matrix assisted laser desorption/ionization-time of flight-time of flight-mass spectrometry (MALDI-TOF-TOF-MS) proteomics analyzer is presented. Protein/peptide samples were separated by capillary high-performance liquid chromatography (cHPLC). The effluents from cHPLC (the first dimension) were continuously transferred into capillary zone electrophoresis (CZE, the second dimension) through a novel valve-free hydrodynamic sampling interface. The CZE effluents were mixed with alpha-cyano-4-hydroxycinnamic acid (CHCA) matrix sheath flow via CE-MALDI interface, and then directly deposited on the MALDI target at a 3 s time-interval for further MS analysis. The high efficiency of the overall system was demonstrated by analysis of proteins in D20 (human hepatocellular carcinoma model in nude mice with high metastatic potential) liver cancer tissue. More than 300 proteins were identified, which proved the system potential for high-throughput analysis and application in proteomics.     

Arsenic speciation by coupling high-performance liquid chromatography with inductively coupled plasma mass spectrometry

Summary An ICP-MS detector in combination with HPLC has been evaluated for the analysis of six arsenic compounds. The influence of the presence of an organic modifier in the mobile phase on arsenic response and the quality parameters of the analysis are discussed. Detection limits for arsenic species under study range from 10 to 30 pg. The determination of arsenic compounds in solutions simulating fish or sediment extracts has been used to demonstrate the applicability of this technique.     

Determination of exemestane and 17-hydroxyexemestane by high-performance liquid chromatography coupled with tandem mass spectrometry and high-resolution mass spectrometry

An approach was developed for determining and confirming the presence of exemestane and its metabolite 17-hydroxyexemestane in urine. It is based on the application of high-performance liquid chromatography coupled with electrospray ionization mass spectrometry (HPLC-MS/MS) and atmospheric pressure chemical ionization high-resolution mass spectrometry (HPLC-HRMS). To detect hydroxyexemestane, the analysis of the hydrolyzed fraction of urine is preferable. The recovery rates of exemestane and 17-hydroxyexemestane were 83 and 91%, respectively. The detection limits were 1 ng/mL for HPLC-MS/MS and 2.5 ng/mL for HPLC-HRMS. In spite of a considerable effect of ionization suppression, the sensitivity and selectivity of the determination are affected by the selection of the optimal detection conditions in HPLC-MS/MS and by the high accuracy of mass determination in mass spectrometry with orbitrap detection, enabling resolution at a level of 5 ppm. The procedures can be used for screening and confirmatory analysis.     

Quasi-linear gradients for capillary liquid chromatography with mass and tandem mass spectrometry

Gradient elution, capillary liquid chromatography mass spectrometry was performed with linear, static gradients constructed by laminar flowing ten, 1.5 microL volume steps of decreasing organic concentration into tubing of small internal diameter. Sample loading, gradient formation, and sample elution were accomplished entirely by means of a commercially available micro-autosampler and single-syringe drive pump. The procedure was simple, fast, stable, and reproducible. Essentially linear gradients were produced without the use of additional valves, mixers, pumps or software. It took less than 10 minutes to form a gradient and less than 30 minutes to construct the set of individual buffer vials. The gradients were shown to be stable to storage. One hour after forming, peak retention times were reproduced to +/-0.5%. Long-term retention time reproducibility was found to vary by +/-2%. Chromatographic resolution was comparable or superior to that obtained by gradient elution with conventional dynamic mixing and split flow. The procedure was adapted with a 'peak parking' method which extended the time for generating peptide fragmentation data up to 10 minutes per peptide with the triple quadruple mass spectrometer. Using this technique, collision data were collected at the 25 femtomole level on nine of ten tryptic peptides in a single run.     

Continuous-flow extraction of organophosphorus pesticides coupled on-line with high-performance liquid chromatography

A continuous-flow extraction system coupled on-line with a high-performance liquid chromatograph with an ultraviolet detector is used to study the extraction of three organophosphorus pesticides (fenthion, azinphos methyl and diazinon) from aqueous samples with n-heptane as the organic solvent. Diazinon was not extracted significantly. The influence on the extraction of different parameters (coil length, flow rate and phase volume ratio) were studied. The calibration graphs are linear for 0.5–7 mg l^?1 and 8–20 mg l^?1 foor azinphos methyl where the percentage extraction (E%) is 90% and 70%, respectively, and up to 4 mg l^?1 for fenthion, where the E% is 33%. The detection limits and the relative standard deviations are 0.04 and 0.09 mg l^?1, and 3.4 and 5.3%, for azinphos methyl fenthion, respectively. Other pesticides and related compounds were found not to interfere. The sample throughput of this method was 15 h^?1.     

Rapid quantification and characterization of soyasaponins by high-performance liquid chromatography coupled with electrospray mass spectrometry

A method using high-performance liquid chromatography (HPLC) with electrospray ionization mass spectrometry (ESI-MS) in the negative mode is presented for the quantification and characterization of different soyasaponins using six authentic soyasaponin standards. This method was successfully applied to the rapid separation of diverse soyasaponins, more than 50, including soyasaponins A in different degrees of acetylation, and soyasaponins B in both their 2,3-dihydro-2,5-dihydroxy-6-methyl-4H-pyran-4-one (DDMP)-conjugated and non-conjugated forms in different samples in one single run for only 30 min. Standard calibration curve was linear over the concentration range of 0.010-1.0 mg/L for each soyasaponin. Within-day and day-to-day relative standard deviations were less than 9.2 and 13.1%, respectively.     

Parallel preparative high-performance liquid chromatography with on-line molecular mass characterization

High-throughput chemistry (HTC) is now an integral part of the lead discovery process in many pharmaceutical and related chemical companies. As this process becomes refined or improved, with the integration of systems with enhanced capabilities, and the requirement for quality compounds of high purity increases, purification is often considered a bottleneck. Although a wide range of purification techniques is available, high-performance liquid chromatography (HPLC) is usually the preferred method of purification to produce high-purity compounds. Parallel preparative HPLC with robust UV-guided fraction collection has been shown to reduce the bottleneck and complement the parallel synthesis systems. However, despite the success of this technique, post-purification analysis of fractions to identify the target compound adds an additional level of complexity. This paper describes the interfacing of the Biotage Parallex with the MUX interface on a single quadrupole mass spectrometer, thus combining robust UV-guided fractionation with on-line MS characterization.