Two-dimensional Liquid Chromatography Method Research Articles

Analysis of pharmaceutical drug oligomers by selective comprehensive two-dimensional liquid chromatography-high resolution mass spectrometry

The presence of oligomeric impurities in drugs is often overlooked and less studied due to conventional small molecule analytical methods which are often not capable of capturing these oligomers. In the present work, the oligomer species of a lipophilic active pharmaceutical ingredient (API) containing an azetidine ring was investigated. No separation was observed by reversed-phase liquid chromatography (RPLC) methods, and only a partial separation of oligomer peaks was achieved by a size exclusion chromatography (SEC) method. To improve the resolution of the different oligomers species and understand the root cause of the formation of the oligomers, a selective comprehensive two-dimensional liquid chromatography (2D-LC) method was developed by coupling SEC to RPLC. The selective comprehensive SEC × RPLC method allowed the separation of 16 species and evidenced four main groups of oligomer impurities. The contour plots of 3 API lots helped to visualize the oligomer profiles and quickly compare the difference between these lots. Finally, the oligomer peaks separated by 2D-LC were identified by high-resolution mass spectrometry (HRMS) using a Q Exactive mass spectrometer. The developed 2D-LC/HRMS workflow provides a fast and generic screening approach to quickly examine and visualize the oligomeric impurities in API materials, and direct the impurity control strategy during process development.

Enrichment and separation of high-polar compounds from Saussurea obvallata using solid-phase extraction combining with offline two-dimensional liquid chromatography.

The high-polar compounds from natural products are often used as medicines due to their good bioactivities. However, owing to the complexity and diversity of their structure, the separation of high-polar compounds is still a challenging work. For this, an efficient method for enrichment and separation of the high-polar compounds from Saussurea obvallata was developed. First, the target compounds were enriched from the total extract using a solid-phase extraction method. An offline two-dimensional liquid chromatography method was used for the separation of pure compounds from the enriched sample. After optimization of chromatographic conditions, high separation selectivity of target compounds was obtained on a polar-modified C18 column and a HILIC XAmide column. Hence, a two-dimensional reversed-phase × hydrophilic interactionliquid chromatography system was constructed and enlarged from the analytical level to the preparative level. In the first dimension, four fractions were obtained on the XCharge C18 column with a recovery rate of 71.2%. In the second-dimension preparation on the XAmide column, eight high-polar compounds with more than 96% purity were isolated. All compounds were isolated from Saussurea obvallata for the first time. The results demonstrated that this developed strategy is effective for preparative-scale isolation of high-polar compounds from natural products.

Determination of intracellular 2-hydroxyglutarate enantiomers using two-dimensional liquid chromatography

In this study, a heart-cutting two-dimensional liquid chromatography (2D-LC) method for the determination of intracellular 2-hydroxyglutarate (2-HG) enantiomers is proposed. D-2-HG is an oncometabolite produced by tumors harboring isocitrate dehydrogenase (IDH) mutations. Hence, achieving accurate quantification of the 2-HG enantiomers in cancer cells is important. In this study, a fluorescence derivatization reagent, 4-nitro-7-piperazino-2,1,3-benzoxadiazole (NBD-PZ), was used. We found that 2-HG was successfully derivatized with NBD-PZ after lactonization. The enantiomers of 2-HG derivatized with NBD-PZ were quantified using the heart-cutting 2D-LC method. NBD-PZ-2-HG was stable for 1 week and was separated from endogenous compounds using an octadecyl silica column before enantiomeric separation on a chiral column. The developed heat-cutting 2D-LC method was validated and successfully applied to cell samples. The amount of D-2-HG and L-2-HG in U937 cells were 9.8 and 8.3 pmol/5.0 × 105 cells, respectively. Furthermore, high amounts of D-2-HG were measured in U937 cells expressing mutant IDH proteins as expected. This method can therefore be applied to further elucidate the role of 2-HG in cancer cells, particularly those harboring isocitrate dehydrogenase mutations.

Profiling of sphingolipids in Caenorhabditis elegans by two-dimensional multiple heart-cut liquid chromatography – mass spectrometry

Sphingolipids exert important functions in cells, ranging from stabilising the cell membrane to bioactive signalling in signal transduction pathways. Changed concentrations of sphingolipids are associated with, among others, neurodegenerative and cardiovascular diseases. In this work, we present a novel two-dimensional liquid chromatography method (2D-LC) coupled to tandem mass spectrometry (MS/MS) for the identification of ceramides, hexosylceramides and sphingomyelins in the model organism Caenorhabditis elegans (C. elegans). The method utilises a multiple heart-cut approach with a hydrophilic interaction liquid chromatography (HILIC) separation in the first dimension. The fractions of the sphingolipid classes were cut out and thereby separated from the abundant glycerolipids, which offers a simplified sample preparation and a high degree of automation as it compensates the alkaline depletion step usually conducted prior to the chromatographic analysis. The fractions were stored in a sample loop and transferred onto the second column with the combination of two six port valves. A reversed phase liquid chromatography was performed as the second dimension and allowed for a separation of the species within a sphingolipid class and according to the fatty acid moiety of the sphingolipid. The segregation of the abundant glycerolipids and the reduced matrix effects allowed for better identification of low abundant species, especially dihydro-sphingolipids with a saturated sphingoid base. In addition, the separation of the three fractions was carried out parallel to the separation and equilibration in the first dimension, which leads to no extension of the analysis time for the 2D-LC compared to the one-dimensional HILIC method. In total 45 sphingolipids were detected in the C. elegans lipid extract and identified via accurate mass and MS/MS fragments.

Online Nanoflow Two-Dimension Comprehensive Active Modulation Reversed Phase–Reversed Phase Liquid Chromatography High-Resolution Mass Spectrometry for Metaproteomics of Environmental and Microbiome Samples

Metaproteomics is a powerful analytical approach that can assess the functional capabilities deployed by microbial communities in both environmental and biomedical microbiome settings. Yet, the mass spectra resulting from these mixed biological communities are challenging to obtain due to the high number of low intensity peak features. The use of multiple dimensions of chromatographic separation prior to mass spectrometry analyses has been applied to proteomics previously but can require increased sampling handling and instrument time. Here, we demonstrate an automated online comprehensive active modulation two-dimensional liquid chromatography method for metaproteome sample analysis. A high pH PLRP-S column was used in the first dimension followed by low pH separation in the second dimension using dual modulating C18 traps and a C18 column. This method increased the number of unique peptides found in ocean metaproteome samples by more than 50% when compared to a one-dimension separation while using the same amount of sample and instrument time.

A comprehensive two-dimensional liquid chromatography method for the simultaneous separation of lipid species and their oxidation products

Lipid oxidation is one of the major causes of food spoilage for lipid-rich foods. In particular, oil-in-water emulsions, like mayonnaises and spreads, are prone to oxidation due to the increased interfacial area that facilitates contact between the lipids and hydrophilic pro-oxidants present in the water phase. Polar, amphiphilic lipid species present at the oil/water interface, like the mono- (MAGs) and di-acylglycerols (DAGs), act as oxidation starters that initiate subsequent oxidation reactions of the non-polar lipids in the oil droplets. A comprehensive two-dimensional liquid chromatography (LC×LC) method with evaporative light-scattering detection (ELSD) was set up to study the composition of the complex mixture of oxidized polar and non-polar lipids. The LC×LC-ELSD method employs size exclusion chromatography (SEC) in the 1D (1st dimension) to separate the various lipid species according to size. In the 2D (2nd dimension), normal-phase liquid chromatography (NPLC) is used to separate the fractions according to their degree of oxidation. The coupling of SEC with NPLC yields a good separation of the oxidized triacylglycerols (TAGs) from the large excess of non-oxidized TAGs. In addition, it allows the isolation of non-oxidized DAGs and MAGs that usually interfere with the detection of a variety of oxidized products that have similar polarities. This method facilitates elucidating how lipid composition affects oxidation kinetics in emulsified foods and will aid in the development of more oxidation-stable products.

Multi-dimensional LC-MS: the next generation characterization of antibody-based therapeutics by unified online bottom-up, middle-up and intact approaches.

Accelerated development of new therapeutics in an increasingly competitive landscape requires the use of high throughput analytical platforms. In addition, the complexity of novel biotherapeutic formats (e.g. fusion proteins, protein-polymer conjugates, co-formulations, etc.) reinforces the need to improve the selectivity and resolution of conventional one-dimensional (1D) liquid chromatography (LC). Liquid chromatography-mass spectrometry (LC-MS)-based technologies such as native LC-MS for intact mass analysis or peptide mapping (also called bottom-up approach)-based multi-attribute methods (MAM) have already demonstrated their potential to complement the conventional analytical toolbox for monoclonal antibody (mAb) characterization. Two-dimensional liquid-chromatography (2D-LC-MS) methods have emerged in the last ten years as promising approaches to address the increasing analytical challenges faced with novel antibody formats. However, off-line sample preparation procedures are still required for conventional 1D and 2D-LC-MS methods for the in-depth variant characterization at the peptide level. Multi-dimensional LC-MS (mD-LC-MS) combine sample preparation and multi-level (i.e. intact, reduced, middle-up and peptide) analysis within the same chromatographic set-up. This review presents an overview of the benefits and limitations of mD-LC-MS approaches in comparison to conventional chromatographic methods (i.e. 1D-LC-UV methods at intact protein level and 1D-LC-MS methods at peptide level). The current analytical trends in antibody characterization by mD-LC-MS approaches, beyond the 2D-LC-MS workhorse, are also reviewed, and our vision on a more integrated multi-level mD-LC-MS characterization platform is shared.

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 Methomyl Residue in Tobacco Samples by Heart-Cutting Two-Dimensional Liquid Chromatography with Tandem Mass Spectrometry.

A novel heart-cutting two-dimensional liquid chromatography coupled with tandem mass spectrometry (2D-LC-MS/MS) was developed for the qualitative and quantitative analysis of methomyl residue in tobacco. Compared to traditional methodologies, fairly high sensitivity and stability were achieved, and the sample procedure was simplified in the two-dimensional liquid chromatography (2D-LC) method. Although methomyl had poor retention performance in most of the reversed-phase liquid chromatography (RPLC) columns, an effective RP/RP strategy was successfully facilitated. An XB-Phenyl column was employed in the first dimension to effectively remove thousands of interference compounds in the matrix. In the second dimension, an ADME column was applied for further separation. After optimization of the separation conditions, a six-way valve was utilized for direct transformation of the target fraction from the 1st column to the 2nd column. A dynamic range of 2.5 ng/mL to 500 ng/mL was achieved with correlation coefficient (r2) greater than 0.9995. The limit of detection and limit of quantification were determined to be 0.69 and 2.30 ng/mL, respectively. The 2D-LC method shows high sensitivity, good reproducibility, and recovery for methomyl in tobacco samples. Therefore, the new method was quite suitable for routine analysis.

Two-dimensional liquid chromatography analysis of all-trans-, 9-cis-, and 13-cis-astaxanthin in raw extracts from Phaffia rhodozyma.

An effective two-dimensional liquid chromatography method has been established for the analysis of all-trans-astaxanthin and its geometric isomers from Phaffia rhodozyma employing a C18 column at the first dimension and a C30 column in the second dimension, connected by a 10-port valve using the photo-diode array detector. The regression equation of astaxanthin calibration curve was established, and the precision and accuracy values were found to be in the range of 0.32-1.14% and 98.21-106.13%, respectively. By using two-dimensional liquid chromatography, it was found that day light, ultrasonic treatment, and heat treatment have significant influence on the content of all-trans-astaxanthin in the extract from P. rhodozyma due to the transformation of all-trans-astaxanthin to cis-astaxanthin. The day light and ultrasonic treatments more likely transform all-trans-astaxanthin to 9-cis-astaxanthin, and the thermal treatment transforms all-trans-astaxanthin to 13-cis-astaxanthin. These results indicate that the two-dimensional liquid chromatography method can facilitate monitoring astaxanthin isomerization in the raw extract from P. rhodozyma. In addition, the study will provide a general reference for monitoring other medicals and bioactive chemicals with geometric isomers.

Multiple heart-cutting mixed-mode chromatography-reversed-phase 2D-liquid chromatography method for separation and mass spectrometric characterization of synthetic oligonucleotides

Until today, ion-pair reversed-phase chromatography is still the dominating method for analytical characterization of synthetic oligonucleotides. Its hyphenation with mass spectrometry, however, has some drawbacks such as ion-suppression in electrospray ionization. To overcome this problem, we present in this work a multiple heart-cutting (MHC) two-dimensional liquid chromatography (2D-LC) method with ultra-violet (UV) and electrospray ionization (ESI) mass spectrometry (MS) detection. A reversed-phase/weak anion-exchange (RP/WAX) stationary phase in the first dimension (1D) provides the selectivity for separation of structurally closely related oligonucleotide sequences and deletions (shortmers), respectively, using a mixed pH/triethylammonium phosphate buffer gradient at constant organic modifier content. Heart cuts of the oligonucleotide peaks are transferred to the second dimension (2D) via a multiple heart-cutting valve which is equipped with two loop decks. The 2D RP column is used for desalting via a diverter valve. Active solvent modulation enables to refocus the oligonucleotide peak into a sharp zone by 2D RP entirely free of non-volatile buffer components and ion-pair agents. Oligonucleotides can thus be sensitively detected by ESI-QTOF-MS under MS-compatible conditions.

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.

Chemometrics in comprehensive two-dimensional liquid chromatography: A study of the data structure and its multilinear behavior

Comprehensive multidimensional chromatographic techniques, such as GC×GC coupled to FID or MS and LC×LC coupled to UV or MS, have gained popularity in recent years. From the analytical perspective, these techniques allow obtaining higher peak capacities and resolution power, as well as adding selectivity from the second orthogonal dimension. From the chemometric point of view, these multidimensional techniques generate highly complex datasets, which present several challenges for their analysis. On the one hand, the selection of the appropriate chemometric data analysis tool requires the understanding of the underlying data structure and its multilinear behavior. On the other hand, peak resolution in complex samples is still a challenge, because of their possible overlapping in one or two chromatographic dimensions despite the increased resolution power.In this work, a comprehensive two-dimensional liquid chromatography method hyphenated simultaneously to PDA and MS detectors was employed for the analysis of a mixture of 31 pharmaceutical compounds. Chemometric evaluation of the obtained two-dimensional chromatograms focuses on two different goals. First, the assessment of the multilinear behavior of the high-dimensional data for each of the two detection modes (LC×LC-UV and LC×LC-MS) and, also, for the multiset data obtained by fusion of the data coming from both detectors. In addition, the chemometric resolution of peaks of overlapping compounds was evaluated using the multivariate curve resolution alternating least squares (MCR-ALS) method. Finally, the advantages of data fusion from UV and MS detectors were discussed, such as the increased ability for compound identification.

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.

Application of the new at-column dilution (ACD) modulator for the two-dimensional RP\xd7HILIC analysis of Buddleja davidii

The focus of this study was the analysis of the complex chemical composition from different parts of Buddleja davidii, whose species are commonly known as ornamental plants and herbal medicines in many countries. As an herbal medicine, it has been utilized for stroke treatments, headache, wound healing, neurological disorder, etc. However, the understanding of its chemical matrices is still insufficient. Therefore, an online two-dimensional reversed phase liquid chromatography x hydrophilic interaction liquid chromatography (RPLCxHILIC) system coupled with mass spectrometry was applied for further detailed investigation of the chemical constituents in Buddleja dividii. In this two-dimensional liquid chromatography (2D-LC) method, a new at-column dilution (ACD) modulator was introduced in the 2D-LC system to solve the incompatibility problem of the mobile phase between two dimensions, which resulted in a 2D-LC analysis with high orthogonality. For the root extract, as one of the analyzed samples, the optimization of the 1D and 2D gradients was carried out carefully. With this new modulator, much better peak separation and better peak shape were achieved compared to two-dimensional liquid chromatography system using a traditional standard (TS) modulator. With a similar approach, the other four parts of Buddleja davidii were well separated. Comparing the different analyzed parts, flowers and leaves showed the most complex profiles. MS and MS/MS data were obtained successfully, which demonstrated the potential of the proposed RPLCxHILIC-MS system in the constituents’ analysis of herbal medicine. However, due to the lack of reported reference information, 24 compounds could be tentatively identified.

Separation of twelve posaconazole related stereoisomers by multiple heart-cutting chiral–chiral two-dimensional liquid chromatography

Posaconazole represents a triazole antifungal agent which is used to treat various fungal infections. It contains four chiral centers leading to 16 stereoisomers. With the convergent synthesis route, only 11 related stereoisomeric impurities may potentially exist in the active pharmaceutical ingredient (API). It is a challenge to separate all the stereoisomers in one run. To address this problem, a multiple heart-cutting chiral–chiral two-dimensional liquid chromatography (2D-LC) method was developed. The multiple heart-cutting 2D-LC separation was implemented on 2D-LC system with three chiral columns with immobilized polysaccharide chiral stationary phases, namely Chiralpak IB, IC and IF3. In the system, the column Chiralpak IB was used as the 1D separation column and IC and IF3 columns were used parallelly for the 2D separation. The twelve stereoisomers were all well separated in one run by the multiple heart-cutting chiral–chiral 2D-LC system.

Application of compressed fluid-based extraction and purification procedures to obtain astaxanthin-enriched extracts from Haematococcus pluvialis and characterization by comprehensive two-dimensional liquid chromatography coupled to mass spectrometry.

The green microalga Haematococcus pluvialis has been widely studied due to its capacity to accumulate great amounts of astaxanthin, a high-value carotenoid with biological activities. In the present work, two green compressed fluid-based processes, pressurized liquid extraction (PLE) and supercritical antisolvent fractionation (SAF), are integrated to obtain an astaxanthin-enriched extract from this microalga. PLE was carried out using pressurized ethanol as solvent, for 20min, at 10MPa, and 50°C as extraction temperature. Subsequently, the obtained extract was processed by SAF to further purify the carotenoid fraction. The SAF process was optimized using a 3-level factorial experimental design and considering three experimental variables: (i) CO2 pressure (10-30MPa), (ii) percentage of water in the PLE extract (20-50%), and (iii) PLE extract/supercritical-CO2 flow rate ratio (0.0125-0.05). Total carotenoid content was evaluated in both extracts and raffinates. Best results were obtained at 30MPa, 0.05 feed/SC-CO2 mass flow rate, and 20% (v/v) of water in the feed solution, achieving values of 120.3mgg-1 carotenoids in extract (in the SAF extract fraction), which were significantly higher than those obtained in the original PLE extract. In parallel, a new fast two-dimensional comprehensive liquid chromatography (LC×LC) method was optimized to get the full carotenoid profile of these extracts in less than 25min. This is the first time that the use of a C30 column is reported in an on-line LC×LC system. Graphical abstract.

Rapid Analysis of Polycyclic Aromatic Hydrocarbons in Water Samples Using an Automated On-line Two-Dimensional Liquid Chromatography

Two-dimensional HPLC (2D-HPLC) recently has received great attention due to providing high resolving power and higher peak capacities than that of 1D-HPLC, especially dealing with a wide spectrum of sample matrices containing several components. In this work, an on-line heart-cutting two-dimensional liquid chromatography (2D-LC) method was developed using monolithic columns coupled with reversed-phase liquid chromatography (RPLC). 2D-HPLC was successfully carried out using affinity-based monolithic columns at first dimension (20 cm × 4.6 mm I.D.) followed by a Pinnacle II PAH column (50 mm × 4.6 mm I.D.) at the second dimension. Furthermore, good linearity was observed for the correlation of the benzo[a]pyrene (BaP) molecule against the peak areas (R2 = 0.994) in the concentration range of 0.01–1.0 ng/mL in 30 min. The limit of detection and the limit of quantification were found to be 4.0 pg/mL and 12.0 pg/mL, respectively. Both the intra-day and inter-day precision at 0.01 and 0.1 ng/mL spiked concentrations were below than 2.35% RSD whereas the means of the recovery data of the BaP from the water samples were found to be in the range of 93.71–98.65%. These results demonstrate that the 2D-HPLC system, developed by the combination of the P(HEMA-MAPA) column and Pinnacle II PAH column, is reliable, stable, and well qualified in the extraction of polycyclic aromatic hydrocarbons from the water samples.

Characterization of Dye Extracts from Historical Cultural-Heritage Objects Using State-of-the-Art Comprehensive Two-Dimensional Liquid Chromatography and Mass Spectrometry with Active Modulation and Optimized Shifting Gradients.

Unbiased characterization of dyes and their degradation products in cultural-heritage objects requires an analytical method which provides universal separation power regardless of dye classes. Dyes are small molecules that vary widely in chemical structure and properties, which renders their characterization by a single method challenging. We have developed a comprehensive two-dimensional liquid chromatography method hyphenated with mass spectrometry and UV–vis detection. We use stationary-phase-assisted modulation to enhance the method in terms of detection limits and solvent compatibility and to reduce the analysis time. The PIOTR program was used to optimize an assembly of shifting second-dimension gradients, which resulted in a high degree of orthogonality (80% in terms of the asterisk concept). The resulting method is universally applicable to all classes of dyes extracted from cultural-heritage objects. Thanks to the high peak capacity and orthogonality, dye components can be separated from chemically similar impurities and degradation products, providing a detailed fingerprint of the dyes mixture in a specific sample. The method was applied to a number of challenging dye extracts from 17th- and 19th-century cultural-heritage objects.

Chemical Profiling and Comparison of Sangju Ganmao Tablet and Its Component Herbs Using Two-Dimensional Liquid Chromatography to Explore Compatibility Mechanism of Herbs.

Sangju Ganmao tablet (SGT), a well-known Chinese patent medicine used to treat cold symptoms, is made from eight herbal medicines. In this study, an off-line hydrophilic interaction × reversed-phase two-dimensional liquid chromatography (HILIC × RP 2D-LC) method was developed to comprehensively separate the chemical constituents of SGT. Through optimization of the experimental conditions, a total of 465 peaks were finally detected in SGT, and the structures of 54 selected compounds were fully identified or tentatively characterized by quadrupole time-of-flight mass spectrometry (qTOF-MS) analysis. The established 2D-LC analysis showed high orthogonality (63.62%) and approximate 11-fold improvement in peak capacity (2399 and 1099, obtained by two calculation methods), in contrast to conventional one-dimensional RPLC separation. The eight component herbs of SGT were also respectively separated by using the 2D-LC system, and we found that a total of 12 peaks detected in SGT were not discovered in any component herbs. These newly generated chemical constituents would benefit better understanding of the compatibility mechanism of the component herbs. The strategy established in this study could be used for systematic chemical comparison of SGT and its component herbs, which contributes to exploration of herbal compatibility mechanism.