Two-dimensional High-performance Liquid Chromatography System Research Articles

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.

Characterization of Acylated Anthocyanins in Red Cabbage via Comprehensive Two-Dimensional High Performance Liquid Chromatography and HPLC-MS

Red cabbage (Brassica oleracea L. Var. Capiata L.) is a fresh edible vegetable abundant in acylated anthocyanins with high antioxidant properties and is a promising resource for the functional food industry. Anthocyanin characterization of red cabbage is not a new topic, but the predominant anthocyanins have been co-eluted using high performance liquid chromatography (HPLC) because of their similar polarities, and it was difficult to achieve a baseline separation of anthocyanins in red cabbage. In the present paper, we optimized the separation of anthocyanins using a two-dimensional HPLC system (2DLC). Using a selective separation column, adjusting the switching time, and optimizing the elution program, seven anthocyanins were separated quickly with a good resolution, and identified via HPLC-mass spectrometry. Therefore, the 2DLC technique proved to be a powerful method of qualitative and quantitative analysis for the anthocyanins of red cabbage. Additionally, this method provides a good reference for the separation of acylated anthocyanins in fruits and vegetables. Practical Applications Red cabbage (Brassica oleracea L. Var. Capiata L.) is a fresh edible vegetable abundant in acylated anthocyanins with high antioxidant properties, and is a promising resource for the functional food industry. This paper describes the development and evaluation of a two-dimensional HPLC system (2DLC) method for the separation of acylated anthocyanins from red cabbage, which provides a good reference for the separation of acylated anthocyanins in fruits and vegetables. And there are many thousands of scientists whose work involve flavonoids, and our work offers a clear example of how new analytical approaches can be employed to increase analytical resolution and therefore enable researchers to clarify previously ambiguous or confounding results. We believe our findings are of interest to readers in food analysis, post-harvest and storage physiology, and to plant biologists that are trying to solve biochemical questions that previous analytical methods have failed to address adequately.

Outlining a Multidimensional Approach for the Analysis of Coffee using HPLC

This study investigated approaches for the profiling of coffee using two multidimensional approaches: (1) a multi-detection process and (2) a multi-separation process employing HPLC. The first approach compared multidetection techniques of conventional High Performance Liquid Chromatography (HPLC) hyphenated with a detector (DPPH•, UV-Vis and MS), and multiplexed mode via HPLC with an Active Flow Technology (AFT) column in Parallel Segmented Flow (PSF) format with DPPH• detection, UV-Vis and MS running simultaneously. Multiplexed HPLCPSF enabled the determination of key chemical entities by reducing the data complexity of the sample whilst obtaining a greater degree of molecule-specific information within a fraction of the time it takes using conventional multi-detection processes. DPPH•, UV-Vis and MS (TIC) were multiplexed for the analysis of espresso coffee and decaffeinated espresso coffee. Up to 20 DPPH• peaks were detected for each sample, and with direct retention time peak matching, 70% of DPPH• peaks gave a UV-Vis response for the espresso coffee and 95% for the decaffeinated espresso coffee. The second approach involved the use of a two-dimensional (2D) HPLC system to expand the separation space and separation power for the analysis of coffee, focusing on the resolution and detection of coeluting and overlapping peaks, which was beyond the limits of conventional HPLC in resolving complex samples. The 2DHPLC analysis resulted with the detection of 176 peaks and a closer observation showed the presence of an additional 17 peaks in a cut section where in 1D mode only one peak was observed.

Development of a two-dimensional high-performance liquid chromatography system coupled with on-line reduction as a new efficient analytical method of 3-nitrobenzanthrone, a potential human carcinogen

3-Nitrobenzanthrone (3-NBA) is an extremely strong mutagen and carcinogen in rats inducing squamous cell carcinoma and adenocarcinoma. We developed a new sensitive analytical method, a two-dimensional HPLC system coupled with on-line reduction, to quantify non-fluorescent 3-NBA as fluorescent 3-aminobenzanthrone (3-ABA). The two-dimensional HPLC system consisted of reversed-phase HPLC and normal-phase HPLC, which were connected with a switch valve. 3-NBA was purified by reversed-phase HPLC and reduced to 3-ABA with a catalyst column, packed with alumina coated with platinum, in ethanol. An alcoholic solvent is necessary for reduction of 3-NBA, but 3-ABA is not fluorescent in the alcoholic solvent. Therefore, 3-ABA was separated from alcohol and impurities by normal-phase HPLC and detected with a fluorescence detector. Extracts from surface soil, airborne particles, classified airborne particles, and incinerator dust were applied to the two-dimensional HPLC system after clean-up with a silica gel column. 3-NBA, detected as 3-ABA, in the extracts was found as a single peak on the chromatograms without any interfering peaks. 3-NBA was detected in 4 incinerator dust samples (n=5). When classified airborne particles, that is, those <1.1, 1.1–2.0, 2.0–3.3, 3.3–7.0, and >7.0μm in size, were applied to the two-dimensional HPLC system after purified using a silica gel column, 3-NBA was detected in those particles with particle sizes <1.1 and 1.1–2.0μm and the particle-size distribution ratios were 84% and 16%, respectively. This is the first report on the particle-size distribution of 3-NBA in airborne particles and the detection of 3-NBA in incinerator dust.

Simultaneous analysis of aspartame and its hydrolysis products of Coca-Cola Zero by on-line postcolumn derivation fluorescence detection and ultraviolet detection coupled two-dimensional high-performance liquid chromatography

An innovative two-dimensional high-performance liquid chromatography system was developed for the simultaneous analysis of aspartame and its hydrolysis products of Coca-Cola Zero. A C8 reversed-phase chromatographic column with ultraviolet detection was used as the first dimension for the determination of aspartame, and a ligand-exchange chromatographic column with on-line postcolumn derivation fluorescence detection was employed as the second dimension for the analysis of amino acid enantiomers. The fluorimetric derivative reagent of amino acid enantiomers was o-phthaldialdehyde. The hydrolysis of aspartame in Coca-Cola Zero was induced by electric-heating or microwave heating. Aspartame was quantified by the matrix matched external standard calibration curve with a linear concentration range of 0–50 μg mL −1 ( r 2 = 0.9984). The limit of detection (LOD) and the limit of quantification (LOQ) were 1.3 μg mL −1 and 4.3 μg mL −1, respectively. The amino acid enantiomers was analyzed by the matrix matched internal standard calibration method ( d-leucine as the internal standard) with a linear concentration range of 0–10 μg mL −1 ( r 2 = 0.9988–0.9997). The LODs and LOQs for l- and d-aspartic acid and l- and d-phenylalanine were 0.16–0.17 μg mL −1 and 0.52–0.55 μg mL −1, respectively, that was 12–13 times more sensitive than ultraviolet detection. The overall analysis accuracy for aspartame and amino acid enantiomers was 90.2–99.2% and 90.4–96.2%, respectively. The overall analysis precision for aspartame and amino acid enantiomers was 0.1–1.7% and 0.5–6.7%, respectively. Generally, the extent of aspartame hydrolysis increases with the increase of electro-thermal temperature, microwave power, and the duration of hydrolysis time. d-aspartic acid and d-phenylalanine can be observed with the electro-thermal racemization at the hydrolysis temperature 120 °C for 1 day and only d-aspartic acid can be observed at the hydrolysis temperature 90 °C for 2 and 3 days. For the microwave induced hydrolysis, only l-aspartic acid was detected at the power 560 W for 1 min and 320 W for 3 min.

Proteomics Analysis Identifies Phosphorylation-dependent α-Synuclein Protein Interactions

Mutations and copy number variation in the SNCA gene encoding the neuronal protein alpha-synuclein have been linked to familial Parkinson disease (Thomas, B., and Beal, M. F. (2007) Parkinson's disease. Hum. Mol. Genet. 16, R183-R194). The carboxyl terminus of alpha-synuclein can be phosphorylated at tyrosine 125 and serine 129, although only a small fraction of the protein is phosphorylated under normal conditions (Okochi, M., Walter, J., Koyama, A., Nakajo, S., Baba, M., Iwatsubo, T., Meijer, L., Kahle, P. J., and Haass, C. (2000) Constitutive phosphorylation of the Parkinson's disease associated alpha-synuclein. J. Biol. Chem. 275, 390-397). Under pathological conditions, such as in Parkinson disease, alpha-synuclein is a major component of Lewy bodies, a pathological hallmark of Parkinson disease, and is mostly phosphorylated at Ser-129 (Anderson, J. P., Walker, D. E., Goldstein, J. M., de Laat, R., Banducci, K., Caccavello, R. J., Barbour, R., Huang, J. P., Kling, K., Lee, M., Diep, L., Keim, P. S., Shen, X. F., Chataway, T., Schlossmacher, M. G., Seubert, P., Schenk, D., Sinha, S., Gai, W. P., and Chilcote, T. J. (2006) Phosphorylation of Ser-129 is the dominant pathological modification of alpha-synuclein in familial and sporadic Lewy body disease. J. Biol. Chem. 281, 29739-29752). Controversy exists over the extent to which phosphorylation of alpha-synuclein and/or the visible protein aggregation in Lewy bodies are steps in disease pathogenesis, are protective, or are neutral markers for the disease process. Here we used the combination of peptide pulldown assays and mass spectrometry to identify and compare protein-protein interactions of phosphorylated and non-phosphorylated alpha-synuclein. We showed that non-phosphorylated alpha-synuclein carboxyl terminus pulled down protein complexes that were highly enriched for mitochondrial electron transport proteins, whereas alpha-synuclein carboxyl terminus phosphorylated on either Ser-129 or Tyr-125 did not. Instead the set of proteins pulled down by phosphorylated alpha-synuclein was highly enriched in certain cytoskeletal proteins, in vesicular trafficking proteins, and in a small number of enzymes involved in protein serine phosphorylation. This targeted comparative proteomics approach for unbiased identification of protein-protein interactions suggests that there are functional consequences when alpha-synuclein is phosphorylated.

Comprehensive two-dimensional high-performance liquid chromatography system with immobilized liposome chromatography column and reversed-phase column for separation of complex traditional Chinese medicine Longdan Xiegan Decoction

A comprehensive two-dimensional HPLC system with an immobilized liposome chromatography (ILC) column in conjunction with an RP column (in tandem) was developed for the screening and analysis of the membrane-permeable compounds in a traditional Chinese medicine prescription Longdan Xiegan Decoction (LXD). More than 50 components in LXD were resolved using the developed separation system. Eight flavonoids and two iridoids were identified interacting with the ILC column; a system that mimics biomembranes. The results show that the developed comprehensive two-dimensional chromatography system can be used for identifying membrane permeable natural products in complex matrixes such as extracts of traditional Chinese medicine prescriptions.

Off-line comprehensive two-dimensional high-performance liquid chromatography system with size exclusion column and reverse phase column for separation of complex traditional Chinese medicine Qingkailing injection

A comprehensive two-dimensional liquid chromatography system was constructed on the combination of size exclusion chromatography (SEC) and reverse phase liquid chromatography (RPLC). The first dimension used a SEC column with 300 mm × 8 mm i.d., packed with Toyopearl HW-40S. The column was eluted with 0.05 mol/l Tris–HCl (pH 6.9) at a flow rate of 0.4 ml/min. The second dimension used a RPLC column with 100 mm × 4.6 mm i.d., which was operated in gradient form at a flow rate of 1.0 ml/min. An automatic switching valve was used to collect the effluent of the SEC column every 3 min, and the effluent was automatically injected into the RPLC column. Mass spectrometer was used for peak identification. This system was used to separate Qingkailing injection, a traditional Chinese medicine (TCM). The result showed that the total peak capacity of this system could reach 1134 and the qualitative analysis of seven chemical components of the Qingkailing injection was accomplished by this system. The results show that comprehensive two-dimensional liquid chromatography system is of great importance and high value in the separation of complex TCM.

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.

Separation for Subsequent Analysis of PCBs, PCDD/Fs, and PAHs According to Aromaticity and Planarity Using a Two-Dimensional HPLC System

In this paper, an automated separation method is described for the subsequent GC/MS analysis of polychlorinated biphenyls (PCBs), polychlorinated dibenzo-p-dioxins and dibenzofurans (PCDD/Fs), polycyclic aromatic compounds (PAHs), and related compounds in complex environmental matrices. The high-performance liquid chromatography (HPLC) method utilizes a coupled column system consisting of a nitrophenylpropylsilica (nitro) column (Nucleosil, 5 μm, 250 × 4.6 mm) and a 2-(1-pyrenyl)ethyldimethylsilylated silica (PYE) column (Cosmosil, 5 μm, 150 × 4.6 mm) and provides five well-defined fractions ready for injection on GC/MS. The fractions are as follow: aliphatic/monocyclic aromatic compounds, mono-tetra-ortho-PCBs, non-ortho-PCBs, PCDD/Fs (tetra-octa-chlorinated), and PAHs. The separation takes less than 40 min and consumes less than 70 mL of solvents. Evaluation of the HPLC system was performed with both environmentally collected samples and standard solution mixtures containing a wide range of PCBs, PCDD/Fs, and PAHs.

Procedure for the gas chromatographic-mass spectrometric confirmation of some exogenous growth-promoting compounds in the urine of cattle

As gas chromatography-mass spectrometry is the most conclusive confirmation technique available today for the detection of ppb levels of anabolics in the urine of cattle, the following procedure was used. The urine is hydrolysed with Helix pomatia intestinal juice, extracted, and the extract cleaned by gel-permeation chromatography or with Extrelut. In one fraction are eluted diethylstilboestrol, dienoestrol, hexoestrol, methyltestosterone, ethinyloestradiol, zeranol and trenbolone. This fraction is injected into a two-dimensional high-performance liquid chromatography system. From the first column, the fraction containing the above-mentioned compounds is transferred to the second column, where a separation into two fractions is obtained. The first fraction contains zeranol and trenbolone, the second fraction the stilbenes, methyltestosterone and ethinyloestradiol. In general, the compounds are derivatized with an N,O-bis (trimethylsilyl)trifluoroacetamide-trimethylchlorosilane mixture to a trimethylsilyl derivative. With stilbene, confirmatory derivatization into heptafluorobutyryl derivatives is necessary. In combination with a Finnigan 4000/INCOS system, a CP-Sil-5 CB and a CP-Sil-19 CB capillary are used for final confirmation. Two capillaries with different polarities are necessary to overcome problems with possible interferences from compounds extracted from the urine. Recoveries at the ppb★ level are better than 80%.