Two-dimensional High-performance Liquid Chromatography Research Articles

Proteomic Analysis of Circulating Extracellular Vesicles Identifies Potential Biomarkers for Lymph Node Metastasis in Oral Tongue Squamous Cell Carcinoma.

Lymph node metastasis is the most reliable indicator of a poor prognosis for patients with oral tongue cancers. Currently, there are no biomarkers to predict whether a cancer will spread in the future if it has not already spread at the time of diagnosis. The aim of this study was to quantitatively profile the proteomes of extracellular vesicles (EVs) isolated from blood samples taken from patients with oral tongue squamous cell carcinoma with and without lymph node involvement and non-cancer controls. EVs were enriched using size exclusion chromatography (SEC) from pooled plasma samples of patients with non-nodal and nodal oral tongue squamous cell carcinoma (OTSCC) and non-cancer controls. Protein cargo was quantitatively profiled using isobaric labelling (iTRAQ) and two-dimensional high-performance liquid chromatography followed by tandem mass spectrometry. We identified 208 EV associated proteins and, after filtering, generated a short list of 136 proteins. Over 85% of the EV-associated proteins were associated with the GO cellular compartment term “extracellular exosome”. Comparisons between non-cancer controls and oral tongue squamous cell carcinoma with and without lymph node involvement revealed 43 unique candidate EV-associated proteins with deregulated expression patterns. The shortlisted EV associated proteins described here may be useful discriminatory biomarkers for differentiating OTSCC with and without nodal disease or non-cancer controls.

Intra-body dynamics of d-serine reflects the origin of kidney diseases

Introductiond-Serine, present only in trace amounts in humans, is now recognized as a biomarker of chronic kidney disease (CKD). CKD is heterogeneous in its original kidney diseases, whose diagnoses require kidney biopsy. In this study, we examined whether the intra-body dynamics of d-serine, indexed by its blood and urinary levels, reflects the origin of kidney diseases.MethodsPatients with six kinds of kidney disease undergoing kidney biopsy were enrolled in a single center. Levels of d- and l-serine were measured using two-dimensional high-performance liquid chromatography. The associations between the origin of kidney diseases and the intra-body dynamics of d-serine were examined using multivariate cluster analyses.ResultsUnlike the non-CKD profile, patients with CKD showed broadly-distributed profiles of intra-body dynamics of d-serine. The plasma level of d-serine plays a key role in the detection of kidney diseases, whereas a combination of plasma and urinary levels of d-serine distinguished the origin of CKD, especially lupus nephritis.ConclusionIntra-body dynamics of d-serine have the potential to predict the origin of kidney diseases. Monitoring of d-serine may guide specific treatments for the origin of kidney diseases.

Determination of tigecycline in human cerebrospinal fluid by two-dimensional liquid chromatography and its clinical application

Objective To establish a two-dimensional high-performance liquid chromatography method for the determination of tigecycline in human cerebrospinal fluid, which can be used for the drug monitoring in patients with intracranial infection. Methods The quantification was carried out by an external standard method. The first-dimension column was a Aston SNX5 phenyl chromatographic column (50 mm×4.6 mm, 5 μm) with ammonium phosphate (pH was adjusted with ammonium hydroxide to 7.5)-methanol (45∶55, V/V) as the mobile phase and the flow rate was 1.2 ml/min. The second-dimension chromatographic column was Aston SC5 C18 (275 mm×4.6 mm, 5 μm), with ammonium phosphate (pH was adjusted with ammonium hydroxide to 7.4)-ammonium phosphate (pH was adjusted with ammonium hydroxide to 3.0)- acetonitrile (30∶50∶20, V/V/V) as the mobile phase and the flow rate was 1.0 ml/min. The detection wavelength was 340 nm. The temperature was 40 ℃ and the injection volume was 200 μl. Results The calibration curve of tigecycline showed good linearity from 64.5 to 1 290.0 ng/ml in human cerebrospinal fluid (r=0.999 8). The RSD of intra and inter-day precision were less than 5.0% with the detection accuracy of 98.80%−106.51%. Conclusion This method is simple, quick, accurate, specific and sensitive. It meets the requirements of tigecycline determination in clinical human cerebrospinal fluid, which offers the individualized therapeutic assurance for patients with intracranial infection.

Determination of phenylalanine enantiomers in the plasma and urine of mammals and ᴅ-amino acid oxidase deficient rodents using two-dimensional high-performance liquid chromatography

A two-dimensional (2D) HPLC system focusing on the determination of phenylalanine (Phe) enantiomers in mammalian physiological fluids has been developed. ᴅ-Phe is indicated to have potential values as a disease biomarker and therapeutic molecule in several neuronal and metabolic disorders, thus the regulation of ᴅ-Phe in mammals is a matter of interest. However, the precise determination of amino acid enantiomers is difficult in complex biological samples, and the development of an analytical method with practically acceptable sensitivity, selectivity and throughput is expected. In the present study, a 2D-HPLC system equipped with a reversed-phase column in the 1st dimension and an enantioselective column in the 2nd dimension has been designed, following the fluorescence derivatization of the target amino acid enantiomers with 4-fluoro-7-nitro-2,1,3-benzoxadiazole (NBD-F). The analytical method was validated using both plasma and urine samples, and successfully applied to human, rat and mouse fluids. Trace levels of ᴅ-Phe were determined in the plasma, and the %ᴅ values were around 0.1% for all species. In the urine, relatively large amounts of ᴅ-Phe were observed, and the %ᴅ values for humans, rats and mice were 3.99, 1.76 and 5.25%, respectively. The relationships between the enzymatic activity of ᴅ-amino acid oxidase (DAO) and the amounts of intrinsic ᴅ-Phe have also been clarified, and high ᴅ-Phe amounts were observed (around 0.3% in the plasma and around 50% in the urine) in the DAO deficient rats and mice.

Application of online two-dimensional high-performance liquid chromatography as purification procedure to determine the origin of 19-norandrosterone in urine by gas chromatography-combustion-isotope ratio mass spectrometry.

19-Norandrosterone (19-NA) is the main metabolite of nandrolone and/or its precursors, which can be found naturally in human urine in trace amount. Gas chromatography-combustion-isotope ratio mass spectrometry (GC-C-IRMS) confirmation procedure can be used to identify a potential exogenous origin of 19-NA in urine sample. Sample purification for GC-C-IRMS analysis is crucial to the whole confirmation procedure because the concentration of 19-NA in the urine to be tested is very low. Online two-dimensional high-performance liquid chromatography (2D-HPLC) clean-up procedure with high separation capacity is used to isolate and enrich 19-NA as a sample pretreatment process. Linearity, lowest detectable concentration, uncertainty, and selectivity of the method are validated according to the World Anti-doping Agency's (WADA) requirement. Isotope fractionation effect was not observed during the 2D-HPLC purification process. The validated method provides a high efficient and convenient confirmation procedure to determine the origin of 19-NA.

Separation of New Coumarin Glycosides from Toddalia asiatica Using Offline Two-Dimensional High-Performance Liquid Chromatography.

Coumarins and flavonoids are the major constituents of Toddalia asiatica. The separation and purification of ingredients from T. asiatica is an important procedure to acquire high-purity compounds for subsequent pharmacological investigation to discover leading compounds. In the present work, an offline two-dimensional high-performance liquid chromatography (HPLC) method was successfully established for the separation of high-purity glycosides from T. asiatica. Based on the separation results obtained with two different chromatographic stationary phases, a phenyl-bonded silica-based reversed-phase column was employed as the first HPLC preparation, and three fractions were obtained from the sample. Then, the fractions were isolated and purified on an octadecyl-bonded silica-based reversed-phase column to obtain high-purity compounds in the second HPLC separation. As a result, three coumarin glycosides, including two undescribed and one known, along with one known flavonoid glycoside with more than 98% purity were isolated from the sample. The structures of the isolated compounds were elucidated on the basis of extensive spectroscopic evidence derived from optical rotation, mass spectrometry, and nuclear magnetic resonance experiments. Two-dimensional HPLC with different stationary phases has the potential to be an efficient method for the separation of high-purity compounds from T. asiatica.

Determination of Risperidone and 9-Hydroxyrisperidone in Human Serum by Heart-Cutting Isocratic Two-Dimensional Liquid Chromatography

This study reports a novel heart-cutting two-dimensional high performance liquid chromatography (2D-LC) method with ultraviolet detection for the determination of risperidone and 9-hydroxyrisperidone in human serum. In this 2D system, a strong cation-exchange column was applied for the first dimensional (1D) separation to isolate the targets from the complex serum matrices, and a middle column was used to transfer the targets from the first dimension to the second dimension (2D) through a six-port valve. A reverse-phase C18 column was selected in the second dimension for further separation. An assistant pump was optimized to prevent the peak dispersion and the loss of resolution. This method has the advantages of high sensitivity by large injection volume, simple one-step sample preparation and short analysis time. A linear range between 5 and 146 ng/mL for risperidone and 7 and 200 ng/mL for 9-hydroxyrisperidone was achieved. All of the results showed that the coupling of ion-exchange with reversed-phase liquid chromatography had powerful separation capabilities for risperidone and 9-hydroxyrisperidone in human serum. The method exhibited good repeatability and recoveries and may be used as a reliable approach for therapeutic drug monitoring in clinical analysis.

Fiber-based HIC capture loop for coupling of protein A and size exclusion chromatography in a two-dimensional separation of monoclonal antibodies

During process development and manufacturing of monoclonal antibodies (mAbs), it is critical to characterize structure-function relationships to properly control the levels of mAb aggregation and potency of the protein product. With two-dimensional high performance liquid chromatography (2D-HPLC) technology, protein A (ProA) affinity chromatography can be used in the first dimension to isolate and measure the concentration of mAb, with the effluent transferred to a second dimension of size exclusion chromatography (SEC) to measure purity (i.e. aggregation). Described here is a 2D-HPLC method for characterizing mAb concentration and aggregation level, combining ProA purification, a novel injector-loop capture step, and aggregation determination by SEC. Unique here, polyester capillary-channeled polymer (C-CP) fibers are packed into PEEK tubing and used as the inter-column injection loop, capturing and releasing the mAbs via a hydrophobic interaction chromatography (HIC) process. The applicability of the HIC capture method was investigated on three ProA columns, including a homemade C-CP fiber format, and commercial POROS® A 20 μm and TSKgel Protein A-5PW columns. The HIC fiber capture loop was compared with standard, open sample loops in terms of solute recoveries, degree of affected aggregation, and chromatographic resolution. Advantages are seen in terms of limiting in-system mAb aggregation due to reduced low-pH solvent exposure, improved 2D chromatographic resolution, better monomer/aggregate ratio fidelity, and enhanced quantitative figures of merit.

The proteome of extracellular vesicles released by clastic cells differs based on their substrate.

Extracellular vesicles (EVs) from osteoclasts are important regulators in intercellular communication. Here, we investigated the proteome of EVs from clastic cells plated on plastic (clasts), bone (osteoclasts) and dentin (odontoclasts) by two-dimensional high performance liquid chromatography mass spectrometry seeking differences attributable to distinct mineralized matrices. A total of 1,952 proteins were identified. Of the 500 most abundant proteins in EVs, osteoclast and odontoclast EVs were 83.3% identical, while clasts shared 70.7% of the proteins with osteoclasts and 74.2% of proteins with odontoclasts. For each protein, the differences between the total ion count values were mapped to an expression ratio histogram (Z-score) in order to detect proteins differentially expressed. Stabilin-1 and macrophage mannose receptor-1 were significantly-enriched in EVs from odontoclasts compared with osteoclasts (Z = 2.45, Z = 3.34) and clasts (Z = 13.86, Z = 1.81) and were abundant in odontoclast EVs. Numerous less abundant proteins were differentially-enriched. Subunits of known protein complexes were abundant in clastic EVs, and were present at levels consistent with them being in assembled protein complexes. These included the proteasome, COP1, COP9, the T complex and a novel sub-complex of vacuolar H+-ATPase (V-ATPase), which included the (pro) renin receptor. The (pro) renin receptor was immunoprecipitated using an anti-E-subunit antibody from detergent-solubilized EVs, supporting the idea that the V-ATPase subunits present were in the same protein complex. We conclude that the protein composition of EVs released by clastic cells changes based on the substrate. Clastic EVs are enriched in various protein complexes including a previously undescribed V-ATPase sub-complex.

Establishment of a microscale peptide prefractionation system based on ultra performance liquid chromatography and the eight-port rotor valve

Liquid chromatography (LC) and mass spectrometry (MS)-based proteomics now allows very deep coverage of proteomes. Two-dimensional high performance liquid chromatography (2-D HPLC) is a useful tool for the proteome analysis of complex biosystem. However, it has drawback of a long running time, and it typically requires peptide amounts in the milligram range, and large volume of collected fractions. In this study, we introduce ultra-performance liquid chromatography (UPLC) and an eight-port rotor valve as a highly efficient and convenient method for a first-dimension separation and collection system. The combination of our UPLC-based fractionation using basic buffers with an online LC-MS/MS provided orthogonal peptide separation and demonstrated the powerful performance for protein identification. Upon applying the novel method to triplicate measurements of a human cell line, we observed excellent quantitative reproducibility between replicates (coefficient of determination R2>0.95) and more than 23.52% peptide identifications over the conventional StageTip approach. The fractionation method described here is flexible, straightforward, and robust, and it enables proteome analysis with minimal sample requirements.

Determination of Trace Amounts of Chiral Amino Acids in Complicated Biological Samples Using Two-Dimensional High-Performance Liquid Chromatography with an Innovative “Shape-Fitting” Peak Identification/Quantification Method

Determination of Trace Amounts of Chiral Amino Acids in Complicated Biological Samples Using Two-Dimensional High-Performance Liquid Chromatography with an Innovative “Shape-Fitting” Peak Identification/Quantification Method

Determination of Ochratoxin A traces in foodstuffs: Comparison of an automated on-line two-dimensional high-performance liquid chromatography and off-line immunoaffinity-high-performance liquid chromatography system

Automated on-line two-dimensional high-performance liquid chromatography method (2D-HPLC) is proposed to determine Ochratoxin A (OTA) in food samples as an alternative to OTA immunoaffinity column (IAC). An on-line 2D-HPLC system is designed for the analysis of OTA using an affinity-based monolithic column in the first dimension and reversed-phase C18 column in the second dimension. Initially, optimal OTA separation efficiency is determined through traditional HPLC system consisting of a P(HEMAPA) monolithic column coupled with HPLC system. Secondly, after providing optimum conditions, OTA determination was investigated through the 2D-HPLC system. According to results, 2D-HPLC system showed good linearity in the range 0.5 to 20 ng/mL with limit of detection (LOD) and limit of quantification (LOQ) values of 21.2 pg/mL and 64.3 pg/mL, respectively. The P(HEMAPA)-4 monolithic column displayed good recovery of OTA ranging from 104.34% to 107.33%. Relative standard deviations (RSD) varied in the range 0.21% to 1.31% thus indicating the efficiency of P(HEMAPA)-4 monolithic column developed for OTA.

D-Serine Induced by Ocular Hypertension is Associated with Retinal Cell Death

Purpose: The purpose of this study is to investigate the role of free D-serine in the death of retinal cells caused by ocular hypertension.Methods: Adult Wistar rats were used as an experimental model of ocular hypertension. Immunohistochemistry was used to identify the retinal sites and expression patterns of D-serine and serine racemase in the rat retina. The concentrations of free D-serine and L-serine in the retina were measured by two-dimensional high-performance liquid chromatography. Retinal cell death was investigated by Immunohistochemistry.Results: D-serine was expressed on the retinal ganglion cell layer in the retinas of rats with ocular hypertension. A serine racemase was specifically expressed in the retinal ganglion cells. The ratio of free D-/L-serine in the retinas with ocular hypertension was higher than that in the retinas with normal tension. Annexing-V-positive cells were observed in the retinal ganglion cell layer in the retinas of the rats with ocular hypertension, and these cells were also co-localized with D-serine expression.Conclusions: We suspect that the up-regulation of serine racemase expression induced by ocular hypertensionleads to an increase in free D-serine converted from free L-serine in retinal ganglion cells and that retinal cell death is associated with D-serine expression.

Complementary enantioselectivity profiles of chiral cinchonan carbamate selectors with distinct carbamate residues and their implementation in enantioselective two-dimensional high-performance liquid chromatography of amino acids

A cardinal requirement for effective 2D-HPLC separations is sufficient complementarity in the retention profiles of first and second dimension separations. It is shown that retention and enantioselectivity of chiral selectors derived from cinchona alkaloids can be conveniently modulated by structural variation of the carbamate residue of the quinine/quinidine carbamate ligand of such chiral stationary phases (CSP). A variety of aliphatic and aromatic residues have been tested in comparison to non-carbamoylated quinine CSP. Various measures of orthogonality have been utilized to derive the CSP that is most complementary to the tert-butylcarbamoylated quinine CSP (tBuCQN CSP), which is commercially available as Chiralpak QN-AX column. It turned out that O-9-(2,6-diisopropylphenylcarbamoyl)-modified quinine is most promising in this respect. Its implementation as a complementary CSP for the separation of amino acids derivatized with Sanger’s reagent (2,4-dinitrophenylated amino acids) in the first dimension combined with a tBuCQN CSP in the second dimension revealed successful enantiomer separations in a comprehensive chiral×chiral 2D-HPLC setup. However, the degree of complementarity could be greatly enhanced when simultaneously the absolute configurations were exchanged from quinine to quinidine in the chiral selector of the first dimension separation resulting in opposite elution orders of the enantiomers in the two dimensions. The advantage of such a chiral×chiral over achiral×chiral 2D-HPLC setup, amongst others, is the perfect compatibility of the mobile phase because in both dimensions the identical eluent can be used.

Enantioselective and simultaneous determination of lactate and 3-hydroxybutyrate in human plasma and urine using a narrow-bore online two-dimensional high-performance liquid chromatography system.

For the enantioselective and simultaneous analysis of lactate and 3-hydroxybutyrate, a validated online two-dimensional high-performance liquid chromatography system using 4-nitro-7-piperazino-2,1,3-benzoxadiazole as a fluorescent derivatization reagent has been developed. For the reversed-phase separation in the first dimension, a Capcell Pak C18 ACR column (1.5×250mm, particle size 3μm) was used, and the target fractions were isolated by their hydrophobicity. In the second dimension, a polysaccharide-coated enantioselective column, Chiralpak AD-H (2.0×250mm, 5μm), was used. The system was validated by the calibration curve, intraday precision, interday precision, and accuracy using standards and real human samples, and satisfactory results were obtained. The present method was applied to human plasma and urine, and in the plasma, trace amounts of d-lactate (8.4μM) and l-3-hydroxybutyrate (1.0μM), besides high levels of l-lactate (860.9μM) and d-3-hydroxybutyrate (59.4μM), were successfully determined. In urine, trace levels of d-lactate (3.7μM), d-3-hydroxybutyrate (2.3μM), and l-3-hydroxybutyrate (3.3μM) in addition to a relatively large amount of l-lactate (15.4μM) were observed. The present online two-dimensional high-performance liquid chromatography system is useful for the simultaneous determination of all the lactate and 3-hydroxybutyrate enantiomers in human physiological fluids, and further clinical applications are ongoing.

Analysis of phosphatidylethanolamine, phosphatidylcholine, and plasmalogen molecular species in food lipids using an improved 2D high-performance liquid chromatography system

Phospholipids are an important class of lipids in cell membranes and food. Several high-performance liquid chromatography (HPLC) methods have been developed to analyze phospholipids at the molecular species level. We developed a two-dimensional HPLC system with a charged aerosol detector and mass spectrometry (MS) to analyze phosphatidylethanolamine (PE), phosphatidylcholine (PC), and their plasmalogens (pls) extracted from food materials. Accordingly, the phospholipid molecular species can be analyzed in a single step despite using smaller samples. We confirmed that chromatogram peaks from soybean lecithin are mostly baseline separated, assigned, and quantified (24 molecular species for PE and 27 for PC). In addition, it was confirmed that chromatograms of lipids extracted from chicken breast meat include plasmalogen peaks. The PE fraction in lipids extracted from chicken breast meat contained 17 types of ethanolamine plasmalogens, corresponding to approximately 57% of the total by weight. The PC fraction contained only four choline plasmalogens, corresponding to approximately 11% of the total weight. The composition of the pls-PC molecular species differed from that of pls-PEs. The polyunsaturated fatty acids connected at the sn-2 positions of the pls-PEs consisted of 20.5% 20:4 fatty acid and were independent of the carbon chain at the sn-1 position. However, the 18:1 fatty acid at the sn-2 position was dependent on the carbon chain at the sn-1 position.

Simultaneous Determination of Polycyclic Aromatic Hydrocarbons and Their Nitro-derivatives in Airborne Particulates by Using Two-dimensional High-performance Liquid Chromatography with On-line Reduction and Fluorescence Detection

Simultaneous Determination of Polycyclic Aromatic Hydrocarbons and Their Nitro-derivatives in Airborne Particulates by Using Two-dimensional High-performance Liquid Chromatography with On-line Reduction and Fluorescence Detection

Two-dimensional chromatographic analysis using three second-dimension columns for continuous comprehensive analysis of intact proteins

We develop a new two-dimensional (2D) high performance liquid chromatography (HPLC) approach for intact protein analysis. Development of 2D HPLC has a bottleneck problem – limited second-dimension (second-D) separation speed. We solve this problem by incorporating multiple second-D columns to allow several second-D separations to be proceeded in parallel. To demonstrate the feasibility of using this approach for comprehensive protein analysis, we select ion-exchange chromatography as the first-dimension and reverse-phase chromatography as the second-D. We incorporate three second-D columns in an innovative way so that three reverse-phase separations can be performed simultaneously. We test this system for separating both standard proteins and E. coli lysates and achieve baseline resolutions for eleven standard proteins and obtain more than 500 peaks for E. coli lysates. This is an indication that the sample complexities are greatly reduced. We see less than 10 bands when each fraction of the second-D effluents are analyzed by sodium dodecyl sulfate – polyacrylamide gel electrophoresis (SDS-PAGE), compared to hundreds of SDS-PAGE bands as the original sample is analyzed. This approach could potentially be an excellent and general tool for protein analysis.

Additional band broadening of peptides in the first size-exclusion chromatographic dimension of an automated stop-flow two-dimensional high performance liquid chromatography

The need to improve the peak capacity of liquid chromatography motivates the development of two-dimensional analysis systems. This paper presented a fully automated stop-flow two-dimensional liquid chromatography system with size exclusion chromatography followed by reversed phase liquid chromatography (SEC×RPLC) to efficiently separate peptides. The effects of different stop-flow operational parameters (stop-flow time, peak parking position, number of stop-flow periods and column temperature) on band broadening in the first dimension (1st D) SEC column were quantitatively evaluated by using commercial small proteins and peptides. Results showed that the effects of peak parking position and the number of stop-flow periods on band broadening were relatively small. Unlike stop-flow analysis of large molecules with a long running time, additional band broadening was evidently observed for small molecule analytes due to the relatively high effective diffusion coefficient (Deff). Therefore, shorter analysis time and lower 1st D column temperature were suggested for analyzing small molecules. The stop-flow two-dimensional liquid chromatography (2D-LC) system was further tested on peanut peptides and an evidently improved resolution was observed for both stop-flow heart-cutting and comprehensive 2D-LC analysis (in spite of additional band broadening in SEC). The stop-flow SEC×RPLC, especially heart-cutting analysis with shorter analysis time and higher 1st D resolution for selected fractions, offers a promising approach for efficient analysis of complex samples.

The use of online heart-cutting high-performance liquid chromatography coupled with linear ion trap mass spectrometry in the identification of impurities in vidarabine monophosphate.

It is difficult to identify unknown impurities in nucleotide analogues by mass spectrometry because mass-spectrometry-incompatible mobile phases need to be used to separate the major ingredient from impurities. In this study, vidarabine monophosphate was selected, and unknown impurities were identified by online heart-cutting two-dimensional high-performance liquid chromatography and linear ion trap mass spectrometry. The one-dimensional reversed-phase column was filled with a mobile phase containing nonvolatile salt. In two-dimensional high-performance liquid chromatography, we used an Acclaim Q1 column with volatile salt, and the detection wavelength was 260nm. The mass spectrum was scanned in positive- and negative-ion mode. The online heart-cutting and online demineralization technique ensured that the mobile phase was compatible with mass spectrometry; seven impurities were identified by MS2 and MS3 fragments. The mass fragmentation patterns of these impurities were investigated. The two isomers were semiprepared and complemented by nuclear magnetic resonance. The results were further compared with those of normal-phase high-performance liquid chromatography with mass spectrometry. The online heart-cutting two-dimensional high-performance liquid chromatography with mass spectrometry was superior in identifying more impurities. The method solves the problem of incompatibility between the mobile phase and mass spectrometry, so it is suitable for identifying unknown impurities. This method may also be used for investigating impurities in other nucleotide analogues.