Two-dimensional Chromatography System Research Articles

Online Two-Dimensional Liquid Chromatography for Determination of Selected Flavonoids in Wine Samples

The development of two-dimensional chromatography systems is crucial in many scientific fields as it provides improved separation efficiency and selectivity of complex mixtures. By separating compounds based on two independent properties, these systems can offer a more detailed understanding of sample composition. This increased level of detail is particularly useful in industries such as pharmaceuticals, environmental analysis, and proteomics, where complex mixtures with similar properties need to be differentiated. A new chromatography system with two dimensions was developed to separate and quantify specific flavonoids in German wine samples. The first dimension used a homemade hydrophilic interaction liquid chromatographic column, while the second dimension used a reversed-phase liquid chromatographic column. This 2D ZIC-HILIC x RP technique was able to address the problem of peak overlapping that commonly occurs in one-dimensional liquid chromatography, especially for natural products. The study presents analytical and statistical data for both techniques, and suggests that the use of two-dimensional chromatography can improve the resolution of conventional one-dimensional liquid chromatography. In a conventional ZIC-HILIC-HPLC system, apigenin and naringenin were detected together but were successfully separated and measured using a 2DLC system. The limits of detection (LOD) for apigenin and naringenin were found to be 0.0705 µg/mL and 0.1300 µg/mL, respectively, while the limits of quantification (LOQ) were 0.2136 µg/mL and 0.3939 µg/mL, respectively. The recovery rate for both compounds was over 99%, with a relative standard deviation (RSD) of less than 1.5%. The linearity range for both compounds was between 0.005 µg/mL to 10 µg/mL, and the correlation coefficient (r2) was determined to be 0.9998. For hesperidin and rutin, the LOD and LOQ were found to be 0.0336 µg/mL and 0.1018 µg/mL, and 0.0120 µg/mL and 0.0363 µg/mL, respectively. The linearity range for both compounds was between 0.005 µg/mL to 12 µg/mL, and the correlation coefficient (r2) was determined to be 0.9999.

1D-Guided Differential Rescaling of a Contour Plot in Comprehensive 2D Gas Chromatography.

A 1D-guided differential rescaling algorithm for a contour plot is developed based on our recently proposed comprehensive two-dimensional gas chromatography (GC × GC) system with a first-dimensional (1D) detector added. Chromatograms obtained from 1D and second-dimensional (2D) detectors are both incorporated during the data processing. As compared to the conventional contour plot methods using only 2D data, our algorithm can significantly improve precision and consistency of GC × GC results in terms of retention times, peak widths, and peak areas or volumes, regardless of modulation time selection, modulation phase shift fluctuations, and modulation duty cycle. The peak identification, quantification, and capacity can therefore be enhanced. Furthermore, the 1D-guided differential rescaling method is shown to better handle the coelution and missing peak issues often encountered in the conventional methods. Finally, the new method exhibits high versatility in 1D and 2D detector selection, which greatly broadens GC × GC utility. Our method can easily be adapted to other two-dimensional chromatography systems that have direct access to 1D chromatograms.

Unveiling Chemical Cues of Insect-Tree and Insect-Insect Interactions for the Eucalyptus Weevil and Its Egg Parasitoid by Multidimensional Gas Chromatographic Methods.

Multidimensional gas chromatography is, presently, an established and powerful analytical tool, due to higher resolving power than the classical 1D chromatographic approaches. Applied to multiple areas, it allows to isolate, detect and identify a larger number of compounds present in complex matrices, even in trace amounts. Research was conducted to determine which compounds, emitted by host plants of the eucalyptus weevil, Gonipterus platensis, might mediate host selection behavior. The identification of a pheromone blend of G. platensis is presented, revealing to be more attractive to weevils of both sexes, than the individual compounds. The volatile organic compounds (VOCs) were collected by headspace solid phase microextraction (HS-SPME), MonoTrapTM disks, and simultaneous distillation-extraction (SDE). Combining one dimensional (1D) and two-dimensional (2D) chromatographic systems—comprehensive and heart-cut two-dimensional gas chromatography (GC×GC and H/C-MD-GC, respectively) with mass spectrometry (MS) and electroantennographic (EAD) detection, enabled the selection and identification of pertinent semiochemicals which were detected by the insect antennal olfactory system. The behavioral effect of a selected blend of compounds was assessed in a two-arm olfactometer with ten parallel walking chambers, coupled to video tracking and data analysis software. An active blend, composed by cis and trans-verbenol, verbenene, myrtenol and trans-pinocarveol was achieved.

Two-dimensional temperature-responsive chromatography using a poly(N-isopropylacrylamide) brush-modified stationary phase for effective therapeutic drug monitoring

Therapeutic drug monitoring (TDM) is an effective pharmacological approach for controlling drug concentration in a patient’s serum. Herein, a new two-dimensional chromatography system was developed using two poly(N-isopropylacrylamide) (PNIPAAm)-modified bead-packed columns for effective and safe drug monitoring. PNIPAAm-modified silica beads were prepared as packing materials using atom transfer radical polymerization of NIPAAm. The increase in the retention times of the drugs requiring TDM with increasing temperature, was attributed to enhanced hydrophobic interactions at elevated temperatures. The drugs and serum proteins were separated on the prepared column at 40 °C using an all-aqueous mobile phase. Differences in the hydrophobic interactions accounted for the elution of the serum proteins and drugs at short and long retention times, respectively, and a primary column was employed to separate the serum proteins and drugs. After eluting the serum proteins from the column, the drug was introduced into the secondary column, leading to a peak of its purified form and enabling determination of the drug concentration. Two-dimensional temperature-responsive chromatography can benefit TDM by allowing the drug concentration in the serum to be measured in all-aqueous mobile phases without sample preparation.

Development of a Method for the Measurement of Human Scent Samples Using Comprehensive Two-Dimensional Gas Chromatography with Mass Detection

Every human body is a source of a unique scent, which can be used for medical or forensic purposes. Human skin scent is a complex mixture of more or less volatile compounds with different chemical and physical properties, which often differ significantly in their concentrations. The most efficient technique for separating such complex samples is comprehensive two-dimensional gas chromatography (GC × GC). This work aimed to find the optimal arrangement of a two-dimensional chromatographic system and define a suitable chromatographic method for non-targeted analysis of human scent samples. Four different chromatographic columns (non-polar Rxi-5MS and TG-5HT, medium polar Rxi-17Sil MS and Rtx-200MS) and their different configurations were tested. The best system was the 30 m primary column Rtx-200MS (with the 2 m pre-column Rtx-200MS) and the 1 m secondary column TG-5HT in a reverse configuration. This system achieved the highest theoretical and conditional peak capacities, optimal resolution, and the lowest number of coelutions.

Recent applications of chemometrics in one- and two-dimensional chromatography.

The proliferation of increasingly more sophisticated analytical separation systems, often incorporating increasingly more powerful detection techniques, such as high‐resolution mass spectrometry, causes an urgent need for highly efficient data‐analysis and optimization strategies. This is especially true for comprehensive two‐dimensional chromatography applied to the separation of very complex samples. In this contribution, the requirement for chemometric tools is explained and the latest developments in approaches for (pre‐)processing and analyzing data arising from one‐ and two‐dimensional chromatography systems are reviewed. The final part of this review focuses on the application of chemometrics for method development and optimization.

An integrated evidence-based targeting strategy for determining combinatorial bioactive ingredients of a compound herbal medicine Qishen Yiqi dripping pills

Ethnopharmacological relevanceQishen Yiqi is a widely used Chinese herbal medicine formula with “qi invigorating and blood activating” property. Its dripping pill preparation (QSYQ) is a commercial herbal medicine approved by the China Food and Drug Administration (CFDA) in 2003 and is extensively used clinically to treat cardiovascular diseases, such as ischemic heart failure and angina pectoris, as well as for the secondary prevention of myocardial infarction. However, the bioactive ingredients of QSYQ remain unclear. As QSYQ is a compound herbal formula, it is of great importance to elucidate its pharmacologically active ingredients and underlying synergetic effects. Aim of the studyThis experimental study was conducted to comprehensively determine the combinatorial bioactive ingredients (CBIs) in QSYQ and to elucidate their potential synergetic effects. The established strategy may shed new light on how to rapidly determine CBIs in complex herbal formulas with holistic properties. Materials and methodsAn integrated evidence-based targeting strategy was introduced and validated to determine CBIs in QSYQ. The strategy included the following steps: (1) Chemical ingredients in QSYQ were analyzed via UPLC-Q-TOF/MS in the negative and positive modes and were identified by comparison with standard compounds and previously reported data. Their potential therapeutic activities were predicted based on the ChEMBL database to preliminarily search for candidate bioactive ingredients, and their combination was defined as the CBIs. (2) The CBIs were directly trapped and prepared from QSYQ with a two-dimensional chromatographic separation system, and the remaining part was defined as the rest ingredients (RIs). (3) As animal and cell models, left anterior descending coronary artery ligation (LAD)-induced heart failure in rats and hypoxia-induced cardiac myocyte injury in H9c2 cells were applied to compare the potency of QSYQ, CBIs and RIs. (4) The synergetic effects on cardiac myocyte protection of multiple ingredients in CBIs were examined in this cell model. Results(1) Forty-three ingredients in QSYQ were identified via UPLC-Q-TOF/MS. Based on evidence-based screening using the ChEMBL database, 24 ingredients were predicted to be bioactive ingredients, and their combination was considered the CBIs. (2) The CBIs and RIs were successfully prepared according to a two-dimensional chromatographic system. The CBIs were directly trapped and knocked out from QSYQ by hydrophilic interaction liquid chromatography coupled with reverse-phase liquid chromatography. The remaining part was used as RIs. (3) The results from pharmacological evaluation revealed that CBIs and QSYQ, but not RIs, significantly prevented myocardium injury; improved the ejection fraction (EF) and fractional shortening (FS); decreased the release of cardiac enzymes, including CK, CK-MB, and LDH; alleviated mitochondrial dysfunction; and protected the cell nucleus number and mitochondrial mass. Furthermore, QSYQ and CBIs possessed similar potency. (4) In hypoxia-stimulated H9c2 cells, CBIs showed far greater potency regarding the protection of cardiac myocyte injury than the individual ingredients in QSYQ, exhibiting obvious synergetic effects. ConclusionsAn integrated evidence-based targeting strategy was successfully established and validated to determine CBIs from QSYQ with excellent efficiency. Importantly, the holistic property of QSYQ was retained in the CBIs. Hence, this study may shed new light on how to rapidly reveal combinatorial bioactive ingredients from complex prescriptions and will be greatly helpful in the establishment of an appropriate approach to quality control for herbal medicines.

Determination of γ-hydroxybutyrate in human urine samples by ion exclusion and ion exchange two-dimensional chromatography system

A two-dimensional ion chromatography system was developed for the determination of γ-hydroxybutyrate (GHB) in human urine samples. Ion exclusion chromatography was used in the first dimensional separation for elimination of urine matrices and detection of GHB above 10mgL−1, ion exchange chromatography was used in the second dimensional separation via column-switching technique for detection of GHB above 0.08mgL−1. Under the optimized chromatographic conditions, the ion exclusion and ion exchange chromatography separation system exhibited satisfactory repeatability (RSD<3.1%, n=6) and good linearity in the range of 50–1000mgL−1 and 0.5–100mgL−1, respectively. By this method, concentrations of GHB in the selected human urine samples were detected in the range of 0–1.57mgL−1. The urine sample containing 0.89mgL−1 GHB was selected to evaluate the accuracy; the spiked recoveries of GHB were 95.9-102.8%. The results showed that the two-dimensional ion chromatography system was convenient and practical for the determination of GHB in human urine samples.

Sample-independent approach to normalize two-dimensional data for orthogonality evaluation using whole separation space scaling

Orthogonality is a key parameter that is used to evaluate the separation power of chromatography-based two-dimensional systems. It is necessary to scale the separation data before the assessment of the orthogonality. Current scaling approaches are sample-dependent, and the extent of the retention space that is converted into a normalized retention space is set according to the retention times of the first and last analytes contained in a unique sample to elute. The presence or absence of a highly retained analyte in a sample can thus significantly influence the amount of information (in terms of the total amount of separation space) contained in the normalized retention space considered for the calculation of the orthogonality. We propose a Whole Separation Space Scaling (WOSEL) approach that accounts for the whole separation space delineated by the analytical method, and not the sample. This approach enables an orthogonality-based evaluation of the efficiency of the analytical system that is independent of the sample selected. The WOSEL method was compared to two currently used orthogonality approaches through the evaluation of in silico-generated chromatograms and real separations of human biofluids and petroleum samples. WOSEL exhibits sample-to-sample stability values of 3.8% on real samples, compared to 7.0% and 10.1% for the two other methods, respectively. Using real analyses, we also demonstrate that some previously developed approaches can provide misleading conclusions on the overall orthogonality of a two-dimensional chromatographic system.

Comprehensive two-dimensional PC-3 prostate cancer cell membrane chromatography for screening anti-tumor components from Radix Sophorae flavescentis.

Radix Sophorae flavescentis is generally used for the treatment of different stages of prostate cancer in China. It has ideal effects when combined with surgical treatment and chemotherapy. However, its active components are still ambiguous. We devised a comprehensive two-dimensional PC-3 prostate cancer cell membrane chromatography system for screening anti-prostate cancer components in Radix Sophorae flavescentis. Gefitinib and dexamethasone were chosen as positive and negative drugs respectively for validation and optimization the selectivity and suitability of the comprehensive two-dimensional chromatographic system. Five compounds, sophocarpine, matrine, oxymatrine, oxysophocarpine, and xanthohumol were found to have significant retention behaviors on the PC-3 cell membrane chromatography and were unambiguously identified by time-of-flight mass spectrometry. Cell proliferation and apoptosis assays confirmed that all five compounds had anti-prostate cancer effects. Matrine and xanthohumol had good inhibitory effects, with half maximal inhibitory concentration values of 0.893 and 0.137mg/mL, respectively. Our comprehensive two-dimensional PC-3 prostate cancer cell membrane chromatographic system promotes the efficient recognition and rapid analysis of drug candidates, and it will be practical for the discovery of prostate cancer drugs from complex traditional Chinese medicines.

Development of APTES-Decorated HepG2 Cancer Stem Cell Membrane Chromatography for Screening Active Components from Salvia miltiorrhiza.

Cell membrane chromatography (CMC) is an ideal method for screening potential active components acting on target cell membranes from a complex system, such as herbal medicines. But due to the decay and falling-off of membranes, the CMC column suffers from short life span and low reproducibility. This has greatly limited the application of this model, especially when the cell materials are hard to obtain. To solve this problem, a novel type of (3-aminopropyl)triethoxysilane (APTES)-decorated silica gel was employed. The silica gel was decorated with aldehydes with the help of APTES, which react with the amino groups on cell membranes to form a covalent bond. In this way, cell membranes were immobilized on the surface of silica gel, so it is not easy for membranes to fall off. According to our investigation, the column life of the APTES-decorated group was prolonged to more than 12 days, while the control group showed a sharp decline in column efficiency in the first 3 days. To verify this model, a novel APTES-decorated HepG2 cancer stem cell membrane chromatography (CSCMC) was established and applied in a comprehensive two-dimensional chromatographic system to screen potential active components in Salvia miltiorrhiza. As a result, tanshinone IIA, cryptotanshinone, and dihydrotanshinone I were retained on this model and proved to be effective on HepG2 cancer stem cells by the following cell proliferation and apoptosis assay, with IC50 of 10.30 μM, 17.85 μM, and 2.53 μM, respectively. This improvement of CMC can significantly prolong its column life span and broaden the range of its application, which is very suitable for making invaluable or hard-to-obtain cell materials, such as stem cells, for specific drug screening.

A new method for the determination of peak distribution across a two-dimensional separation space for the identification of optimal column combinations.

For the identification of the optimal column combinations, a comparative orthogonality study of single columns and columns coupled in series for the first dimension of a microscale two-dimensional liquid chromatographic approach was performed. In total, eight columns or column combinations were chosen. For the assessment of the optimal column combination, the orthogonality value as well as the peak distributions across the first and second dimension was used. In total, three different methods of orthogonality calculation, namely the Convex Hull, Bin Counting, and Asterisk methods, were compared. Unfortunately, the first two methods do not provide any information of peak distribution. The third method provides this important information, but is not optimal when only a limited number of components are used for method development. Therefore, a new concept for peak distribution assessment across the separation space of two-dimensional chromatographic systems and clustering detection was developed. It could be shown that the Bin Counting method in combination with additionally calculated histograms for the respective dimensions is well suited for the evaluation of orthogonality and peak clustering. The newly developed method could be used generally in the assessment of 2D separations. Graphical Abstract ᅟ.

Micro Two-Dimensional Thin-Layer Chromatography and Chemometric Analysis of Essential Oils from Selected Mentha Species and Its Application in Herbal Fingerprinting

Micro two-dimensional thin-layer chromatographic systems (micro-2D-TLCs) were optimized using some popular nonaqueous mobile phases on 5 cm × 5 cm silica plates. The optimal 2D TLC systems were chosen based on correlations of RF values vs. mobile phases concentrations performed for 1D separations. Finally plates were developed using the following mobile phases: in the first direction – ethyl acetate/toluene (5/95; v/v) and in the second direction – ethyl acetate/n-heptane (15/85; v/v). Eleven essential oils obtained by hydrodistillation in Deryng apparatus from various mint specimens were separated and then 2D thin-layer chromatograms were densitometrically scanned at the wavelength of 254 nm. Digital images (exported from densitometer software as ASCII files) were processed using ImageJ free software, and then some chemometric parameters were calculated (determination coefficient, congruence coefficient, Euclidean’s distance, Manhattan’s distance, Chebyshev’s distance) and additionally, principal component analysis (PCA) was performed to determine the goodness of matching for individual essential oils. The chemometric analysis allows one to identify individual essential oils and the examined plant materials.

Simultaneous achiral-chiral analysis of pharmaceutical compounds using two-dimensional reversed phase liquid chromatography-supercritical fluid chromatography

A new interface was designed to enable the coupling of reversed phase liquid chromatography (RPLC) and supercritical fluid chromatography (SFC). This online two-dimensional chromatographic system utilizing RPLC in the first dimension and SFC in the second was developed to achieve simultaneous achiral and chiral analysis of pharmaceutical compounds. The interface consists of an eight-port, dual-position switching valve with small volume C-18 trapping columns. The peaks of interest eluting from the first RPLC dimension column were effectively focused as sharp concentration pulses on small volume C-18 trapping column/s and then injected onto the second dimension SFC column. The first dimension RPLC separation provides the achiral purity result, and the second dimension SFC separation provides the chiral purity result (enantiomeric excess). The results are quantitative enabling simultaneous achiral, chiral analysis of compounds. The interface design and proof of concept demonstration are presented. Additionally, comparative studies to conventional SFC and case studies of the applications of 2D LC–SFC in pharmaceutical analysis is presented.

Study on the performance of different types of three-dimensional chromatographic systems

The maximum achievable performance of possible types of three-dimensional chromatographic systems (LC×LC×LC) has been investigated. The Pareto-optimization approach was applied to establish a trade-off between three main objectives (total peak capacity, analysis time and dilution of the sample) and Pareto-front values were obtained. The performances of xLC×xLC×xLC (three-dimensional separation in space), tLC×tLC×tLC (three-dimensional separation in time) and the hybrid xLC×xLC×tLC system were compared mutually and with two-dimensional chromatographic systems. It was found that xLC×xLC×xLC performs best in terms of maximum achievable peak capacity in shortest analysis time. Based on current thin-layer-chromatography performance it should be possible to obtain a peak capacity of 50,000 within 20min. If contemporary column-packing standards can be upheld the achievable limit is approximately 50% higher. However, in an xLC×xLC×xLC chromatographic system analytes remain in the separation domain after the analysis, which complicates the detection. Use of an xLC×xLC×tLC system with elution in the last dimension alleviates the detection problem. The maximum achievable peak capacity in the same analysis time is lower for xLC×xLC×tLC than for xLC×xLC×xLC. Using the same (reasonable) length of the separation domain (e.g. a cube 200×200×200mm) for both systems, it is possible to achieve peak capacities of 78,000 for xLC×xLC×tLC operated in the gradient mode, which is twice higher than for an xLC×xLC×xLC system. A three-dimensional (three-column) time-based tLC×tLC×tLC system does not greatly improve the performance of tLC×tLC in terms of (maximum) peak capacity and (minimum) analysis time. Dilution factors in tLC×tLC×tLC are very high. Decreasing the dilution has a detrimental influence on the peak capacity. The trade-off between these objectives is of crucial importance. The influence of several parameters (length of the separation domain, particle size, etc.) on the performance of chromatographic systems was investigated, optimal ranges were found.

Pareto-optimality study into the comparison of the separation potential of comprehensive two-dimensional liquid chromatography in the column and spatial modes

The expected performance of spatial (“flat-bed”) two-dimensional liquid chromatography (xLC×xLC) has been calculated using the Pareto-optimality strategy. This approach allowed different objectives (total peak capacity, total analysis time, and total dilution) to be considered simultaneously and to establish optimal parameters (pressure drop, particle size, bed length, and initial spot size). The performance of spatial two-dimensional chromatographic systems was compared with that of conventional on-line, real-time two-dimensional column-liquid-chromatography systems (tLC×tLC). The potential gain in peak capacity and/or analysis time of the spatial configuration was confirmed. By restricting the spatial parameters to realistic chromatographic conditions (limiting the stress, as counterbalance for the pressure drop through the sorbent bed, to 2500kg) it was found that xLC×xLC is attractive for very fast analysis of complex samples, rather than for extremely efficient separations. For example, a peak capacity of 780 may be achieved in only 2.7min using a 100×100mm sorbent bed of a quality currently encountered thin-layer chromatography. Furthermore, if beds can be packed as efficiently as contemporary columns, the predicted peak capacity increases to around 1000, corresponding to a peak-production rate of about 6.3peaks/s. Possibilities to boost the performance of xLC×xLC further are briefly discussed. Unless we can overcome the severe stress requirements of high-performance xLC×xLC, conventional tLC×tLC may be more amenable to very complex separations, thanks to the very high peak capacities. However, tLC×tLC separations will require long analysis times (e.g. 10,000 peaks in 37h, corresponding to 0.075peaks/s at a pressure drop of 40MPa). The best trade-off between total peak capacity, total analysis time, and total dilution under restricted (realistic) conditions was obtained using high pressures, small chromatographic beds, small particles, and relatively large sample spots.

Rapid T cell–based identification of human tumor tissue antigens by automated two-dimensional protein fractionation

Identifying the antigens that have the potential to trigger endogenous antitumor responses in an individual cancer patient is likely to enhance the efficacy of cancer immunotherapy, but current methodologies do not efficiently identify such antigens. This study describes what we believe to be a new method of comprehensively identifying candidate tissue antigens that spontaneously cause T cell responses in disease situations. We used the newly developed automated, two-dimensional chromatography system PF2D to fractionate the proteome of human tumor tissues and tested protein fractions for recognition by preexisting tumor-specific CD4+ Th cells and CTLs. Applying this method using mice transgenic for a TCR that recognizes an OVA peptide presented by MHC class I, we demonstrated efficient separation, processing, and cross-presentation to CD8+ T cells by DCs of OVA expressed by the OVA-transfected mouse lymphoma RMA-OVA. Applying this method to human tumor tissues, we identified MUC1 and EGFR as tumor-associated antigens selectively recognized by T cells in patients with head and neck cancer. Finally, in an exemplary patient with a malignant brain tumor, we detected CD4+ and CD8+ T cell responses against two novel antigens, transthyretin and calgranulin B/S100A9, which were expressed in tumor and endothelial cells. The immunogenicity of these antigens was confirmed in 4 of 10 other brain tumor patients. This fast and inexpensive method therefore appears suitable for identifying candidate T cell antigens in various disease situations, such as autoimmune and malignant diseases, without being restricted to expression by a certain cell type or HLA allele.

Development of two-dimensional micro strong cation exchange liquid chromatography/reversed phase pressurized capillary electrochromatography and its application on &lt;I&gt;Cortex Phellodendri&lt;/I&gt; extract

Pressurized capillary electrochromatography (pCEC) combines capillary electrophoresis (CE) with high performance liquid chromatography (HPLC), resulting in high separation efficiency, high selectivity, high resolution and fast speed. In addition, it provides a solvent gradient capability. Based on pCEC, a two-dimensional chromatography was constructed using micro strong cation exchange liquid chromatography and reversed phase pressurized capillary electrochromatography (micro-SCXLC/RP-pCEC) for the analysis of Cortex Phellodendri extract. A sample was separated by the first dimensional SCX column with a linear salt gradient elution into 11 plugs. Each plug was collected and injected into the second dimensional RP-pCEC column with a linear gradient for further separation. Compared with one-dimensional liquid chromatography, it has higher resolution and larger peak capacity. The two-dimensional chromatographic system is suitable for the separation and analysis of complex samples.

Setting up a 2D‐LC/MS/MS method for the rapid quantitation of the prostanoid metabolites 6‐oxo‐PGF1α and TXB2 as markers for hemostasis assessment

6-oxo-PGF(1alpha) and TXB(2) are the metabolites of the prostanglandin PGI(2) and of the thromboxane TXA(2), respectively. PGI(2) and TXA(2) are arachidonic acid-derived compounds which regulate the blood hemostasis. Their quick metabolism leads to the 6-oxo-PGF(1alpha) and TXB(2) metabolites in plasma. In order to study on a large base the external factors influencing the hemostatic conditions, there is a need for a fast and reliable assay for quantitating these metabolites. Some methods have been published for the analysis of the arachidonic acid-derived compounds and some are dealing with mass spectrometry but nonspecifically centered on these specific compounds with a fast and cheap protocol, amenable for large-scale studies. Here we describe an analytical strategy that incorporates a two-dimensional chromatography running coupled to tandem mass spectrometry that minimizes the sample preparation and addresses the presence of the TXB(2) anomers for a robust quantitation measurement. After a protein precipitation, 100 microl of the supernatant (corresponding to 50 microl of the original plasma) was injected in a two-dimensional chromatographic system which operates an on-line clean-up and a subsequent chromatographic separation of the targeted analytes with a limit of quantitation (LOQ) of 22 pg/ml for 6-oxo-PGF(1alpha), and and a LOQ of 25 pg/ml for TXB(2).

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.