On-line Two-dimensional Liquid Chromatography Research Articles

Profiles of a broad spectrum of epigenetic DNA modifications in normal and malignant human cell lines: Proliferation rate is not the major factor responsible for the 5-hydroxymethyl-2'-deoxycytidine level in cultured cancerous cell lines.

Active demethylation of 5-methylcytosine moiety in DNA occurs by its sequential oxidation to 5-hydroxymethylcytosine, 5-formylcytosine and 5-carboxycytosine, catalysed by enzymes of the Ten-Eleven Translocation family proteins (TETs 1, 2 and 3). Here we analyzed for the first time all the intermediate products of DNA demethylation pathway in the form of deoxynucleosides (5-methyl-2′-deoxycytidine, 5-(hydroxymethyl)-2′-deoxycytidine, 5-formyl-2′-deoxycytidine and 5-carboxy-2′-deoxycytidine as well as 5-(hydroxymethyl)-2′-deoxyuridine) using automated isotope-dilution online two-dimensional ultra-performance liquid chromatography with tandem mass spectrometry. DNA was isolated from human malignant cell lines of colon adenocarcinoma (HCT 116), melanoma (Me45), myelogenous leukemia bone marrow blasts (K562), EBV-positive Burkitt’s lymphoma lymphoblasts (Raji), EBV-negative Burkitt’s lymphoma lymphoblasts (male-CA46 and female-ST486), as well as normal neonatal dermal fibroblasts (NHDF-Neo). The expression levels of TET1, TET2, TET3, SMUG1, and TDG genes were also assayed by RT-qPCR. Our results show a global erasure of 5-hydroxymethyl-2′-deoxycytidine and 5-carboxy-2′-deoxycytidine in DNA of cultured cells compared with DNA from primary malignant tissue. Moreover, malignant cells in culture have a quite different DNA epigenetic profile than cultured normal cells, and different types of malignant cells display different and characteristic profiles of DNA epigenetic marks. Similar analyses of a broader spectrum of epigenetic modifications, not restricted to 5-methyl-2′-deoxycytidine, could lead to better understanding of the mechanism(s) responsible for emergence of different types of cancer cells.

The Combination Process for Preparative Separation and Purification of Paclitaxel and 10-Deacetylbaccatin III Using Diaion® Hp-20 Followed by Hydrophilic Interaction Based Solid Phase Extraction.

There is no other naturally occurring defense agent against cancer that has a stronger effect than paclitaxel, commonly known under the brand name of Taxol®. The major drawback for the more widespread use of paclitaxel and its precious precursor, 10-deacetylbaccatin III (10-DAB III), is that they require large-scale extraction from different parts of yew trees (Taxus species), cell cultures, taxane-producing endophytic fungi, and Corylus species. In our previous work, a novel online two-dimensional heart-cut liquid chromatography process using hydrophilic interaction/ reversed-phase chromatography was used to introduce a semi-preparative treatment for the separation of polar (10-deacetylbaccatin III) and non-polar (paclitaxel) taxanes from Taxus baccata L. In this work, a combination of the absorbent (Diaion®HP-20) and a silica based solid phase extraction is utilized as a new, efficient, and cost effective method for large-scale production of taxanes. This process avoids the technical problem of two-dimensional preparative liquid chromatography. The first stage of the process involves discarding co-extractive polar compounds including chlorophylls and pigments using a non-polar synthetic hydrophobic absorbent, Diaion®HP-20. Extract was then loaded on to a silica based hydrophilic interaction solid phase extraction (silica 40-60 micron). Taxanes was eluted using a mixture of water and methanol at the optimized ratio of 70:30. Finally, the fraction containing taxanes was applied to semi-preparative reversed phase HPLC. The results revealed that using this procedure, paclitaxel and 10-DAB III could be obtained at 8 and 3 times more, respectively than by the traditional method of extraction.

Comprehensive Dual Liquid Chromatography with Quadruple Mass Spectrometry (LC1MS2 × LC1MS2 = LC2MS4) for Analysis of Parinari Curatellifolia and Other Seed Oil Triacylglycerols.

Online two-dimensional (2D) comprehensive liquid chromatography (LC × LC) has become increasingly popular. Most LC × LC separations employ one or more detectors at the outlet of the second dimension, 2D, with very short runs to avoid undersampling. We used six detectors, including dual parallel mass spectrometry (LC1MS2), for detection of the first dimension, 1D. We made an argentation (silver-ion) UHPLC column from a strong cation exchange column for 2D, coupled with UV and LC1MS2 detection. LC1MS2 in 1D combined with LC1MS2 in 2D, plus five other detectors, constituted LC2MS4 in a comprehensive LC1MS2 × LC1MS2 2D-LC separation. Electrospray ionization (ESI) high resolution accurate mass (HRAM) mass spectrometry (MS) and atmospheric pressure chemical ionization (APCI) MS were used in parallel for 1D detection, while atmospheric pressure photoionization (APPI) MS and ESI-MS were used for detection of 2D. The LC1MS2 used for 1D allowed quantification of triacylglycerol (TAG) molecular species of Parinari curatellifolia and other seed oils, while the 2D allowed isomers of TAG containing 18:3 fatty acyl chains as well as TAG regioisomers to be separated and identified. The LC1MS2 in 1D allowed identification of oxo-TAG species by HRAM MS and quantification of 806.3 ± 1.3 and 1101 ± 22 μg/g of α- and γ- tocopherols, respectively, in P. curatellifolia by APCI-MS. It is now feasible to use silver-ion UHPLC as the 2D separation in LC × LC and to use multiple mass spectrometers across both dimensions to perform conventional quantitative analysis and to take advantage of the newest LC × LC separation technology to identify isomers that are otherwise difficult to separate.

Comprehensive two dimensional liquid chromatography as analytical strategy for pharmaceutical analysis

Comprehensive on-line two-dimensional liquid chromatography (LCxLC) is expected to generate impressive peak capacities, which makes it a method of choice for the analysis of complex samples such as pharmaceuticals. A comparative study of different sets of chromatographic conditions including stationary phase, pH additive and organic modifier was carried out with two real pharmaceutical samples in order to find out the best analytical conditions for implementation of one or several generic on-line LCxLC separations. Our choice was based on the evaluation of both degree of orthogonality and practical sample peak capacity under linear gradient conditions. The potential of 190 combinations of chromatographic systems was compared. A set of 3 RPLCxRPLC configurations was found to be very attractive for both samples and in good agreement with the findings of a previous study carried out with 17 model compounds, thereby supporting the idea of using generic LCxLC conditions in the pharmaceutical area. The three selected 2D-systems were implemented for the on-line RPLCxRPLC-UV/MS analysis of two pharmaceutical samples. It was shown, for each sample, that these 2D-systems were able to generate an effective peak capacity close to 1000 in less than 50min. For each sample, baseline separation was obtained for every known compound and furthermore a large number of unknown impurities could also be separated and identified. Finally, in the proposed conditions, the total number of compounds detected was significantly improved from one RPLC separation to one RPLCxRPLC separation. Only a small additional gain was observed by performing a second RPLCxRPLC separation or even a third one.

Increasing Flexibility in Two-Dimensional Liquid Chromatography by Pulsed Elution of the First Dimension: A Proof of Concept.

This work demonstrates the development of an online two-dimensional liquid chromatography (2D-LC) method where the first dimension column is eluted by a sequence of pulses of increasing eluotropic strength generated by the LC pumps (pulsed-elution 2D-LC). Between the pulses, the first dimension is kept in a no-elution state using low eluent strength. The eluate from the first dimension is actively modulated using trap columns and subsequently analyzed in the second dimension. We demonstrate that by tuning the length and eluotropic strength of the pulses, peaks with retention factors in water, kw, above 150 can be manipulated to elute in 3-4 pulses. The no-elution state can be kept for 1-10 min with only minor changes as to which and how many pulses the peaks elute in. Pulsed-elution 2D-LC combined with active modulation tackles three of the main challenges encountered in 2D-LC and specifically online comprehensive 2D-LC: undersampling, difficulties in refocusing, and lack of flexibility in the selection of column dimensions and flow rates because the two dimensions constrain each other. The pulsed-elution 2D-LC was applied for the analysis of a basic fraction of vacuum gas oil. Peak capacity was 4018 for a 540 min analysis and 4610 for a 1040 min analysis.

Monolithic stationary phases with a longitudinal gradient of porosity.

The duration of the hypercrosslinking reaction has been used to control the extent of small pores formation in polymer-based monolithic stationary phases. Segments of five columns hypercrosslinked for 30-360min were coupled via zero-volume unions to prepare columns with segmented porosity gradients. The steepness of the porosity gradient affected column efficiency, mass transfer resistance, and separation of both small-molecule alkylbenzenes and high-molar-mass polystyrene standards. In addition, the segmented column with the steepest porosity gradient was prepared as a single column with a continuous porosity gradient. The steepness of porosity gradient in this type column was tuned. Compared to a completely hypercrosslinked column, the column with the shallower gradient produced comparable size-exclusion separation of polystyrene standards but allowed higher column permeability. The completely hypercrosslinked column and the column with porosity gradient were successfully coupled in online two-dimensional liquid chromatography of polymers.

Accurate, Direct, and High-Throughput Analyses of a Broad Spectrum of Endogenously Generated DNA Base Modifications with Isotope-Dilution Two-Dimensional Ultraperformance Liquid Chromatography with Tandem Mass Spectrometry: Possible Clinical Implication.

Our hereby presented methodology is suitable for reliable assessment of the most common unavoidable DNA modifications which arise as a product of fundamental metabolic processes. 8-Oxoguanine, one of the oxidatively modified DNA bases, is a typical biomarker of oxidative stress. A noncanonical base, uracil, may be also present in small quantities in DNA. A set of ten-eleven translocation (TET) proteins are involved in oxidation of 5-methylcytosine to 5-hydroxymethylcytosine which can be further oxidized to 5-formylcytosine and 5-carboxycytosine. 5-Hydroxymethyluracil may be formed in deamination reaction of 5-hydroxymethylcytosine or can be also generated by TET enzymes. All of the aforementioned modifications seem to play some regulatory roles. We applied isotope-dilution automated online two-dimensional ultraperformance liquid chromatography with tandem mass spectrometry (2D-UPLC-MS/MS) for direct measurement of the 5-methyl-2'-deoxycytidine, 5-(hydroxymethyl)-2'-deoxycytidine, 5-formyl-2'-deoxycytidine, 5-carboxy-2'-deoxycytidine, 5-(hydroxymethyl)-2'-deoxyuridine, 2'-deoxyuridine, and 8-oxo-2'-deoxyguanosine. Analyses of DNA extracted from matched human samples showed that the 5-(hydroxymethyl)-2'-deoxycytidine level was 5-fold lower in colorectal carcinoma tumor in comparison with the normal one from the tumor's margin; also 5-formyl-2'-deoxycytidine and 5-carboxy-2'-deoxycytidine were lower in colorectal carcinoma tissue (ca. 2.5- and 3.5-fold, respectively). No such differences was found for 2'-deoxyuridine and 5-(hydroxymethyl)-2'-deoxyuridine. The presented methodology is suitable for fast, accurate, and complex evaluation of an array of endogenously generated DNA deoxynucleosides modifications. This novel technique could be used for monitoring of cancer and other diseases related to oxidative stress, aberrant metabolism, and environmental exposure. Furthermore, the fully automated two-dimensional separation is extremely useful for analysis of material containing a considerable amount of coeluting interferents with mass-spectrometry-based methods.

Vitamin C enhances substantially formation of 5-hydroxymethyluracil in cellular DNA

The most plausible mechanism behind active demethylation of 5-methylcytosine involves TET proteins which participate in oxidation of 5-methylcytosine to 5-hydroxymethylcytosine; the latter is further oxidized to 5-formylcytosine and 5-carboxycytosine. 5-Hydroxymethyluracil can be also generated from thymine in a TET-catalyzed process. Ascorbate was previously demonstrated to enhance generation of 5-hydroxymethylcytosine in cultured cells. The aim of this study was to determine the levels of the abovementioned TET-mediated oxidation products of 5-methylcytosine and thymine after addition of ascorbate, using an isotope-dilution automated online two-dimensional ultra-performance liquid chromatography with electrospray ionization tandem mass spectrometry. Intracellular concentration of ascorbate was determined by means of ultra-performance liquid chromatography with UV detection. Irrespective of its concentration in culture medium (10–100µM) and inside the cell, ascorbate stimulated a moderate (2- to 3-fold) albeit persistent (up to 96-h) increase in the level of 5-hydroxymethylcytosine. However, exposure of cells to higher concentrations of ascorbate (100µM or 1mM) stimulated a substantial increase in 5-formylcytosine and 5-carboxycytosine levels. Moreover, for the first time we demonstrated a spectacular (up to 18.5-fold) increase in 5-hydroxymethyluracil content what, in turn, suggests that TET enzymes contributed to the presence of the modification in cellular DNA. These findings suggest that physiological concentrations of ascorbate in human serum (10–100µM) are sufficient to maintain a stable level of 5-hydroxymethylcytosine in cellular DNA. However, markedly higher concentrations of ascorbate (ca. 100µM in the cell milieu or ca. 1mM inside the cell) were needed to obtain a sustained increase in 5-formylcytosine, 5-carboxycytosine and 5-hydroxymethyluracil levels. Such feedback to elevated concentrations of ascorbate may reflect adaptation of the cell to environmental conditions.

Overview of online two-dimensional liquid chromatography based on cell membrane chromatography for screening target components from traditional Chinese medicines.

Cell membrane chromatography is a simple, specific, and time-saving technique for studying drug-receptor interactions, screening of active components from complex mixtures, and quality control of traditional Chinese medicines. However, the short column life, low sensitivity, low column efficiency (so cannot resolve satisfactorily mixture of compounds), low peak capacity, and inefficient in structure identification were bottleneck in its application. Combinations of cell membrane chromatography with multidimensional chromatography such as two-dimensional liquid chromatography and high sensitivity detectors like mass have significantly reduced many of the above-mentioned shortcomings. This paper provides an overview of the current advances in online two-dimensional-based cell membrane chromatography for screening target components from traditional Chinese medicines with particular emphasis on the instrumentation, preparation of cell membrane stationary phase, advantages, and disadvantages compared to alternative approaches. The last section of the review summarizes the applications of the online two-dimensional high-performance liquid chromatography based cell membrane chromatography reported since its emergence to date (2010-June 2016).

An optimised method for the proteomic profiling of full thickness human skin.

BackgroundThe skin is the largest organ of the human body and is the first line barrier defence against trauma, microbial infiltration and radiation. Skin diseases can be a result of multi-systemic disease or an isolated condition. Due to its proteolysis resistant properties there are relatively few human skin proteomic datasets published compared with other human organs or body fluids. Skin is a challenging tissue to analyse using traditional proteomic techniques due to its high lipid content, insolubility and extensive cross-linking of proteins. This can complicate the isolation and digestion of proteins for analysis using mass spectrometry techniques.ResultsWe have optimised a sample preparation procedure to improve solubilisation and mass spectral compatibility of full thickness skin samples. Using this technique, we were able to obtain data for the proteome profile of full thickness human skin using on-line two-dimensional liquid chromatography, followed by ultra-high definition label-free mass spectrometry analysis (UDMSE). We were able to identify in excess of 2000 proteins from a full thickness skin sample.ConclusionsThe adoption of on-line fractionation and optimised acquisition protocols utilising ion mobility separation (IMS) technology has significantly increased the scope for protein identifications ten-fold.Electronic supplementary materialThe online version of this article (doi:10.1186/s12575-016-0045-y) contains supplementary material, which is available to authorized users.

Array-Based Online Two Dimensional Liquid Chromatography System Applied to Effective Depletion of High-Abundance Proteins in Human Plasma.

In this work, an array-based online two-dimensional liquid chromatography (2D-LC) system was constructed for protein separation and effective depletion of high-abundance proteins in human plasma. This system employed a strong anion exchange column in the first dimension and eight reversed-phase liquid chromatographic columns in the second dimension. All the protein components in the first dimension were enriched on the trapping columns, simultaneously back-flushed and concurrently separated in the second dimension. LC eluents were then collected on 96-well plates for further analysis. Compared with common 2D-LC system, this system showed an 8-fold increase in throughput and convenient utilization of stop-flow mode for sample separation. The RSD of retention time and peak area were separately below 0.51% and 8%. Recovery rates of four standard proteins were all above 95%. This array-based 2D-LC system was subsequently applied to the analysis of proteins in human plasma. The eluents containing high-abundance proteins were rapidly located according to the results of bicinchoninic acid assay. In all, with the effective depletion of 84 high-abundance proteins, a total of 1332 proteins were identified through our system. The dynamic range of the identified protein concentrations covered 9 orders of magnitude, ranging from 41 g/L level for HSA down to 0.01 ng/mL level for the low-abundance proteins.

On-line Fast Purification of Three Kinds of Active Proteins from Egg White with Two-dimensional Liquid Chromatography Using a Single Column

With the development of biotechnology and life science, both recombinant protein drug productions and proteomics research depend largely on fast and efficient protein separation technology. Analysis of complex samples has put forward higher and higher requirements to separation science. Therefore, developing new separation material, separation mode and more sensitive detection method should be one of the effective ways to solve these problems. Based on a novel strong cation exchange/hydrophobic interaction chromatography (SCX/HIC) dual-function column, which displays HIC character in a high salt concentration, and IEC character in a low salt concentration in mobile phase employed and can be employed to separate proteins with SCX and HIC modes, respectively, a new on-line two-dimensional liquid chromatography technology with a single column (2DLC-1C) was developed. An equipment with automatic control system called as "ChromatoExpert" for pro- cessing fast protein separation online 2DLC-1C was constructed, which has multiple functions, such as on-line fraction col- lection from the first separation mode and quantitatively sample re-injection into the second separation mode, on-line buffer exchange, etc. Eight kinds of protein standards were separated using SCX/HIC 2DLC-1C. The unretained and uncompletely separated proteins in SCX mode were collected online and could be separated completely with each other by online re-injection and second dimensional separation of HIC mode. As a result, these eight kinds of proteins can be separated with- in 2 h. It also was applied to purify three kinds of active intact proteins from egg white successfully. The results indicated that lysozyme, ovotransferrin and ovalbumin from egg white could be purified just within 70 min by online 2DLC-1C. The purity of these proteins obtained was 95%, 93% and 97%, respectively. The new online 2DLC-1C technology can simplify the puri- fication process and lower the separation cost. It is easy to realize automation and scale-up to preparative scale. Therefore, this new technology has a broad application prospect in the fast separation and preparation of active protein. Keywords online 2DLC-1C; protein; purification; egg white

Two‐dimensional liquid chromatography and its application in traditional Chinese medicine analysis and metabonomic investigation

Two-dimensional liquid chromatography has become an attractive analytical tool for the separation of complex samples due to its enhanced selectivity, peak capacity, and resolution compared with one-dimensional liquid chromatography. Recently, more attention has been drawn on the application of this separation technique in studies concerning traditional Chinese medicines, metabonomes, proteomes, and other complex mixtures. In this review, we aim to examine the application of two-dimensional liquid chromatography in traditional Chinese medicine analysis and metabonomic investigation. The classification and evaluation indexes were first introduced. Then, various switching methods were summarized when used in an on-line two-dimensional liquid chromatography system. Finally, the applications of this separation technique in traditional Chinese medicine analysis and metabonomic investigation were discussed on the basis of specific studies.

Tissue-Specific Differences in DNA Modifications (5-Hydroxymethylcytosine, 5-Formylcytosine, 5-Carboxylcytosine and 5-Hydroxymethyluracil) and Their Interrelationships.

BackgroundReplication-independent active/enzymatic demethylation may be an important process in the functioning of somatic cells. The most plausible mechanisms of active 5-methylcytosine demethylation, leading to activation of previously silenced genes, involve ten-eleven translocation (TET) proteins that participate in oxidation of 5-methylcytosine to 5-hydroxymethylcytosine which can be further oxidized to 5-formylcytosine and 5-carboxylcytosine. Recently, 5-hydroxymethylcytosine was demonstrated to be a relatively stable modification, and the previously observed substantial differences in the level of this modification in various murine tissues were shown to depend mostly on cell proliferation rate. Some experimental evidence supports the hypothesis that 5-hydroxymethyluracil may be also generated by TET enzymes and has epigenetic functions.ResultsUsing an isotope-dilution automated online two-dimensional ultra-performance liquid chromatography with tandem mass spectrometry, we have analyzed, for the first time, all the products of active DNA demethylation pathway: 5-methyl-2′-deoxycytidine, 5-hydroxymethyl-2′-deoxycytidine, 5-formyl-2′-deoxycytidine and 5-carboxyl-2′-deoxycytidine, as well as 5-hydroxymethyl-2′-deoxyuridine, in DNA isolated from various rat and porcine tissues. A strong significant inverse linear correlation was found between the proliferation rate of cells and the global level of 5-hydroxymethyl-2′-deoxycytidine in both porcine (R2 = 0.88) and rat tissues (R2 = 0.83); no such relationship was observed for 5-formyl-2′-deoxycytidine and 5-carboxyl-2′-deoxycytidine. Moreover, a substrate-product correlation was demonstrated for the two consecutive steps of iterative oxidation pathway: between 5-hydroxymethyl-2′-deoxycytidine and its product 5-formyl-2′-deoxycytidine, as well as between 5-formyl-2′-deoxycytidine and 5-carboxyl-2′-deoxycytidine (R2 = 0.60 and R2 = 0.71, respectively).ConclusionsGood correlations within the substrate-product sets of iterative oxidation pathway may suggest that a part of 5-formyl-2′-deoxycytidine and/or 5-carboxyl-2′-deoxycytidine can be directly linked to a small portion of 5-hydroxymethyl-2′-deoxycytidine which defines the active demethylation process.

Optimization of conditions in on-line comprehensive two-dimensional reversed phase liquid chromatography. Experimental comparison with one-dimensional reversed phase liquid chromatography for the separation of peptides

Comprehensive on-line two-dimensional liquid chromatography is an attractive technique to enhance peak capacity and deal with complex samples. Yet, its optimization is not straightforward and requires to take into account a huge number of parameters to finally obtain a good compromise between different criteria which are often conflicting. In this study, we propose a procedure for RPLC×RPLC separations able to define, for a given analysis time, the optimized LC×LC parameters for achieving the best compromise between high peak capacity and low dilution. This procedure makes use of both a prediction tool based on theoretical relationships and a Pareto-optimality approach to optimize these two criteria. The possibility to split the flow-rate of the first dimension before the interface is taken into account as well as the focusing effect in the second dimension. This optimization procedure is applied to the prediction of optimized conditions for the separation of peptides. The need for very short and efficient columns (typically 30mm as column length and 1.3μm as particle diameter) in the second dimension is clearly established whatever the geometry of the first dimension column. Furthermore it is shown that several sets of quite different conditions in the first dimension can lead to similar results and hence that additional quality descriptors are required to make a decision. RPLC×RPLC separations of a protein digest were carried out and the obtained results were found in very good accordance with the predicted ones. Our results were compared to the top results reported in the literature for 1D-RPLC separations of peptides. More than 4-fold increase in peak capacity (5100 vs 1150) at 200min has been obtained. Furthermore a 40-fold gain in analysis time (1h vs 40h) was obtained for achieving 1800 as effective peak capacity which is close to the highest peak capacity reported in 1D-RPLC for the separation of peptides.

A reversed-phase/hydrophilic interaction mixed-mode C18-Diol stationary phase for multiple applications

A mixed-mode chromatographic packing material, C18 and diol groups modified silica (C18-Diol), was prepared with controllable hydrophobicity and hydrophilicity. It demonstrated excellent aqueous compatibility and stability in aqueous mobile phase; compared to the traditional C18 column, improved peak shape of basic analytes was also obtained. Additionally, it exhibited both reversed-phase liquid chromatographic (RPLC) and hydrophilic interaction chromatographic (HILIC) performance; the analyte separation scope was thus enlarged, demonstrated by simultaneous separation of twenty acids, bases and neutrals. More interestingly, a novel on-line two-dimensional liquid chromatography on the single column (2D-LC-1C) was established by modifying the high performance liquid chromatographic instrument only with the addition of an extra six-port two-position valve. The early co-eluted components of the extract of Lonicera japonica on the 1st-dimension (RPLC) were collected for the online re-injection to the 2nd-dimension (HILIC) by conveniently varying the mobile phase components. Six more peaks were obtained. The established system was simple, easy operation and low cost, which had advantages in analyzing complicated samples.

Characterization of sodium carboxymethyl cellulose by comprehensive two-dimensional liquid chromatography

Two series of sodium carboxymethyl celluloses (NaCMC) with average degrees of substitution (DS) ranging from 0.45 to 1.55 were synthesized from low molecular mass Avicel cellulose (Avicel samples) and from high molecular mass cotton linters (BWL samples). The samples were characterized by online two-dimensional liquid chromatography using gradient liquid adsorption chromatography in the first and size exclusion chromatography (SEC) in the second dimension. This method allows the simultaneous determination of the chemical composition (DS) and the molar mass distribution within the individual samples. Moreover information was obtained on the dependence of the elution volume in gradient chromatography on molar mass. As expected, evidence for a stronger influence of molar mass on gradient elution volume was found for the low molecular mass NaCMC as compared to the high molecular mass BWL samples. Finally the applicability of the method for the simultaneous separation of blends heterogeneous with respect to chemical composition (DS) and molar mass was demonstrated. Such blends cannot be efficiently separated by either SEC or gradient chromatography alone, nor by simply combining the results of both methods. Only the complete two-dimensional chromatogram can reveal the complexity of such blends, since it reveals the correlations between molar mass and chemical composition.

Simultaneous determination of vitamins A, D3 and E in infant formula and adult nutritions by online two-dimensional liquid chromatography

A rapid method for the simultaneous determination of vitamins A, D3 and E in infant formula and adult nutritions has been developed using online two-dimensional liquid chromatography (2D-LC). First of all, C8 and polar embedded C18 columns were chosen as the first and second dimensional column respectively according to hydrophobic-subtraction model, which constituted excellent orthogonal separation system. The detection wavelengths were set at 263 nm for vitamin D3, 296 nm for vitamin E and 325 nm for vitamin A. The purification of vitamin D3 and quantifications of vitamins A and E were completed simultaneously in the first dimensional separation using the left pump of Dual Gradient LC (DGLC) with methanol, acetonitrile and water as mobile phases. The heart-cutting time window of vitamin D3 was confirmed according to the retention time of vitamin D3 in the first dimensional separation. The elute from the first dimensional column (1-D column) which contained vitamin D3 was collected by a 500 µL sample loop and then taken into the second dimensional column (2-D column) by the right pump of DGLC with methanol, acetonitrile and water as mobile phases. The quantification of vitamin D3 was performed in the second dimensional separation with vitamin D2 as internal standard. At last, this method was applied for the analysis of the three vitamins in milk powder, cheese and yogurt. The injected sample solution with no further purification was pre-treated by hot-saponification using 1. 25 kg/L KOH solution and extracted by petroleum ether solvent. The recoveries of vitamin D3 spiked in all samples were 75.50%-85.00%. There was no statistically significant difference for the results between this method and standard method through t-test. The results indicate that vitamins A, D3 and E in infant formula and adult fortified dairy can be determined rapidly and accurately with this method.

A not-stop-flow online normal-/reversed-phase two-dimensional liquid chromatography–quadrupole time-of-flight mass spectrometry method for comprehensive lipid profiling of human plasma from atherosclerosis patients

A not-stop-flow online two-dimensional (2D) liquid chromatography (LC) method was developed for comprehensive lipid profiling by coupling normal- and reversed-phase LC with quadrupole time-of-flight mass spectrometry (QToF-MS), which was then applied to separate and identify the lipid species in plasma, making its merits in quality and quantity of the detection of lipids. Total 540 endogenous lipid species from 17 classes were determined in human plasma, and the differences in lipid metabolism products in human plasma between atherosclerosis patients and control subjects were explored in detail. The limit of detections (LODs) of 19 validation standards could all reach ng/mL magnitude, and the RSDs of peak area and retention time ranged 0.4–8.0% and 0.010–0.47%, respectively. In addition, a pair of isomers, galactosylceramides (GalC) and glucosylceramides (GluC), was successfully separated, showing that only the levels of GalC in atherosclerosis patients were significantly increasing, rather than GluC, compared with the controls (controls vs. patients: the ratio was 1.5–2.8-fold increasing). It would be helpful to the further research of the atherosclerosis.

Analysis of Glycated Albumin by On-Line Two-Dimensional Liquid Chromatography Mass Spectrometry

Nonenzymatic reaction between glucose and proteins leads to glycated proteins. There is evidence to suggest that protein glycation may be involved in the long-term complications of diabetes mellitus. Knowledge about which proteins are glycated and the sites of glycation may provide new insight in understanding the mechanisms of diabetic complications. Therefore, the development of methods for comprehensive identification of glycated proteins is crucial to elucidate their biological effect. In this report, boronate affinity chromatography (BAC) was coupled on-line to LC–MS for comprehensive analysis of the in vitro glycated bovine serum albumin (BSA). Additionally, in comparison to analysis by conventional collision induced dissociation (CID) MS2 and neutral loss-triggered MS3 (NLMS3), multistage activation (MSA) significantly improved the number of identified glycated peptides. In summary, the established on-line BAC LC–MS approach identified 37 glycation sites in glycated BSA.