Purification of lignans from Fructus Arctii using off‐line two‐dimensional supercritical fluid chromatography/reversed‐phase liquid chromatography

As a common traditional Chinese medicine, Fructus Arctii has important clinical medical values. Its main components are lignans, which are difficult to separate and analyze because of the complex composition, similar chemical structures, and close properties. In this study, an off‐line two‐dimensional supercritical fluid chromatography/reversed‐phase liquid chromatography method, as well as an effective sample pretreatment method based on hydrophilic interaction chromatography material, was developed to enrich the minor lignan fractions and obtain high‐purity compounds. In total, 12 high‐purity compounds were isolated from Fructus Arctii . Their structures were identified by using high‐resolution mass spectrometry and nuclear magnetic resonance spectroscopy, which showed that all were lignans and that most of them were isomers. The results demonstrated the effective off‐line two‐dimensional supercritical fluid chromatography/reversed‐phase liquid chromatography method for the purification of lignans from Fructus Arctii . The separation protocol established here will be beneficial for the separation of complex samples from other kinds of natural products.     

Optimizing gradient conditions in online comprehensive two‐dimensional reversed‐phase liquid chromatography by use of the linear solvent strength model

The linear solvent strength model was used to predict coverage in online comprehensive two‐dimensional reversed‐phase liquid chromatography. The prediction model uses a parallelogram to describe the separation space covered with peaks in a system with limited orthogonality. The corners of the parallelogram are assumed to behave like chromatographic peaks and the position of these pseudo‐compounds was predicted. A mix of 25 polycyclic aromatic compounds were used as a test. The precision of the prediction, span 0–25, was tested by varying input parameters, and was found to be acceptable with root mean square errors of 3. The accuracy of the prediction was assessed by comparing with the experimental coverages. Less than half of experimental coverages were outside prediction ± 1 × root mean square error and none outside prediction ± 2 × root mean square error. Accuracy was lower when retention factors were low, or when gradient conditions affected parameters not included in the model, e.g. second dimension gradient time affects the second dimension equilibration time. The concept shows promise as a tool for gradient optimization in online comprehensive two‐dimensional liquid chromatography, as it mitigates the tedious registration and modeling of all sample constituents, a circumstance that is particularly appealing when dealing with complex samples.     

Off‐line comprehensive two‐dimensional reversed‐phase countercurrent chromatography with high‐performance liquid chromatography: Orthogonality in separation of Polygonum cuspidatum Sieb. et Zucc

Off‐line comprehensive two‐dimensional reversed‐phase countercurrent chromatography with high‐performance liquid chromatography was investigated in separation of crude ethanol extract from traditional Chinese medicinal herb Polygonum cuspidatum Sieb. et Zucc. Two‐dimensional contour plots for countercurrent chromatography with high‐performance liquid chromatography was obtained after comprehensive separation was completed. Total peak capacity was evaluated and approximately 810 peaks were obtained through a comprehensive two‐dimensional separation. A highly orthogonality of 52.23% and a large separation space occupancy of 88.86% were achieved. Meanwhile, it was found that several components could be well separated by countercurrent chromatography while they could not be separated by high‐performance liquid chromatography, and vice versa, which further indicated the orthogonality of the two separation methods. The off‐line comprehensive two‐dimensional countercurrent chromatography with high‐performance liquid chromatography provided a promising and powerful method for separation of complex natural products.     

Comprehensive two‐dimensional monolithic liquid chromatography of polar compounds

Two‐dimensional liquid chromatography largely increases the number of separated compounds in a single run, theoretically up to the product of the peaks separated in each dimension on the columns with different selectivities. On‐line coupling of a reversed‐phase column with an aqueous normal‐phase (hydrophilic interaction liquid chromatography) column yields orthogonal systems with high peak capacities. Fast on‐line two‐dimensional liquid chromatography needs a capillary or micro‐bore column providing low‐volume effluent fractions transferred to a short efficient second‐dimension column for separation at a high mobile phase flow rate. We prepared polymethacrylate zwitterionic monolithic micro‐columns in fused silica capillaries with structurally different dimethacrylate cross‐linkers. The columns provide dual retention mechanism (hydrophilic interaction and reversed‐phase). Setting the mobile phase composition allows adjusting the separation selectivity for various polar substance classes. Coupling on‐line an organic polymer monolithic capillary column in the first dimension with a short silica‐based monolithic column in the second dimension provides two‐dimensional liquid chromatography systems with high peak capacities. The silica monolithic C₁₈ columns provide higher separation efficiency than the particle‐packed columns at the flow rates as high as 5 mL/min used in the second dimension. Decreasing the diameter of the silica monolithic columns allows using a higher flow rate at the maximum operation pressure and lower fraction volumes transferred from the first, hydrophilic interaction dimension, into the second, reversed‐phase mode, avoiding the mobile phase compatibility issues, improving the resolution, increasing the peak capacity, and the peak production rate.     

Evaluation of matrix effect in one‐dimensional and comprehensive two‐dimensional liquid chromatography for the determination of the phenolic fraction in extra virgin olive oils

Olea europaea, meaning “European olive,” is a small tree belonging to the family Oleaceae, occurring in the Mediterranean Basin. Olive oil is an essential component of a balanced diet because of its nutritional value. Among micronutrients, phenolic compounds did show important beneficial effects for human health. The majority of the research studies on the phenol content are carried out by liquid chromatography combined to photodiode array and/or mass spectrometry detection; however, because of matrix complexity, one‐dimensional liquid chromatography cannot be sometimes sufficient to obtain rewarding separations, requiring more advanced analytical techniques. In this work, comprehensive two‐dimensional liquid chromatography, incorporating RP‐Amide and C18 stationary phases, in the first and second dimension, respectively, both under reversed phase conditions, was investigated for the determination of the phenolic fraction in extra virgin olive oil samples. As far as detection is concerned, triple quadrupole mass spectrometry was employed under multi reaction monitoring mode offering superior selectivity and sensitivity. The reduction of matrix effects, when using comprehensive two‐dimensional liquid chromatography with respect to conventional one‐dimensional liquid chromatography, was assessed by comparing the slopes of calibration curves built from standard solutions and spiked olive oil samples.     

Application of two‐dimensional liquid chromatography in the separation of traditional Chinese medicine

Traditional Chinese medicines have been widely used to prevent and cure diseases for thousands of years. For the purpose of better understanding the extremely complicated traditional Chinese medicines, powerful separation techniques are essential. Two‐dimensional liquid chromatography has been proven to be more powerful for the separation of complex traditional Chinese medicines due to its enhanced peak capacity and resolution compared with one‐dimensional liquid chromatography. Enormous efforts have been made on the coupling of independent separation mechanisms to improve the resolving power for complex traditional Chinese medicine samples, including the development and introduction of novel stationary phases. This review aims to give an overview on the applications of two‐dimensional liquid chromatography in traditional Chinese medicine research since 2008, including comprehensive two‐dimensional liquid chromatography, heart‐cutting two‐dimensional liquid chromatography both in on‐line, and off‐line mode. Different couplings of separation modes were respectively discussed based on specific studies, with emphasis on the applications of novel stationary phases in the two‐dimensional liquid chromatography.     

Development of an improved online comprehensive hydrophilic interaction chromatography × reversed‐phase ultra‐high‐pressure liquid chromatography platform for complex multiclass polyphenolic sample analysis

In this study, an improved online comprehensive two‐dimensional liquid chromatography platform coupled to tandem mass spectrometry was developed for the analysis of complex polyphenolic samples. A narrowbore hydrophilic interaction chromatography column (150 × 2.0 mm, 3.0 μm, cross‐linked diol) was employed in the first dimension, while a reversed‐phase column based on monodisperse sub‐2 μm fully porous particles (50 × 3.0 mm, 1.9 μm d.p.) with high surface area (410 m²/g) was employed in the second dimension. The combination of a trapping column modulation interface with the high retentive fully porous monodisperse reversed‐phase column in the second dimension resulted in higher peak capacity values (1146 versus 867), increased sensitivity, sharper and more symmetrical peaks in comparison with a conventional loop‐based method, with the same analysis time (70 min). The system was challenged against a complex polyphenolic extract of a typical Italian apple cultivar, enabling the simultaneous separation of multiple polyphenolic classes, including oligomeric procyanidins, up to degree of polymerization of 10. Hyphenation with an ion trap time‐of‐flight mass spectrometer led to the tentative identification of 121 analytes, showing how this platform could be a powerful analytical tool for the accurate profiling of complex polyphenolic samples.     

Characterization and quantification of triacylglycerols in peanut oil by off‐line comprehensive two‐dimensional liquid chromatography coupled with atmospheric pressure chemical ionization mass spectrometry

The complexity of natural triacylglycerols (TAGs) in various edible oils is prodigious due to the hundreds of set is of TAG compositions, which makes the identification of TAGs quite difficult. In this investigation, the off‐line 2D system coupling of nonaqueous RP and silver‐ion HPLC with atmospheric pressure chemical ionization MS detection has been applied to the identification and quantification of TAGs in peanut oil. The method was successful in the separation of a high number of TAG solutes, and the TAG structures were evaluated by analyzing their atmospheric pressure chemical ionization mass spectra information. HPLC and MS conditions have been optimized and the fragmentation mechanisms of isomers have been validated. In addition, an internal standard approach has been developed for TAG quantification. Then this system was applied in peanut oil samples and there was a total of 48 TAGs including regioisomers that have been determined and quantified.     

Chemical profiling of bioactive constituents in hop cones and pellets extracts by online comprehensive two‐dimensional liquid chromatography with tandem mass spectrometry and direct infusion Fourier transform ion cyclotron resonance mass spectrometry

Humulus lupulus L. (hop) is highly interesting from a nutraceutical perspective. The hop phytocomplex contains a wide range of bioactive metabolites, and its characterization is challenging. To tackle such a task, for the first time we applied and compared a combined approach consisting of online comprehensive two‐dimensional liquid chromatography with tandem mass spectrometry and direct infusion Fourier transform ion cyclotron mass spectrometry. A reversed phase × reversed phase approach with a shifted gradient in the second dimension ensured selectivity and two‐dimensional space coverage. Hyphenation with an ion trap time‐of‐flight analyzer led to the identification of 83 compounds in 70 min, comprising a novel quercetin derivative and six unknown bitter acids. On the other hand, the direct infusion method was able to identify 40 analytes (except isomers) with high mass accuracy (≤ 0.1 ppm) in less than 1 min analysis time. The developed approach can be used in a complementary way, combining the separation capability and high informative spectra of two‐dimensional liquid chromatography tandem mass spectrometry with the ultra‐high mass accuracy of direct infusion, for potential compound discovery or the accurate profiling of bioactive compounds in different hop cultivars as well as for monitoring processing and storage of hop‐based products.     

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 × 250 mm, particle size 3 μm) 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 × 250 mm, 5 μm), 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 (8.4 μM) and l‐ 3‐hydroxybutyrate (1.0 μM), besides high levels of l‐ lactate (860.9 μM) and d‐ 3‐hydroxybutyrate (59.4 μM), were successfully determined. In urine, trace levels of d‐ lactate (3.7 μM), d‐ 3‐hydroxybutyrate (2.3 μM), and l‐ 3‐hydroxybutyrate (3.3 μM) in addition to a relatively large amount of l‐ lactate (15.4 μM) 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.     

Assessing the detectability of antioxidants in two‐dimensional high‐performance liquid chromatography

This paper explores the analytical figures of merit of two‐dimensional high‐performance liquid chromatography for the separation of antioxidant standards. The cumulative two‐dimensional high‐performance liquid chromatography peak area was calculated for 11 antioxidants by two different methods—the areas reported by the control software and by fitting the data with a Gaussian model; these methods were evaluated for precision and sensitivity. Both methods demonstrated excellent precision in regards to retention time in the second dimension (%RSD below 1.16%) and cumulative second dimension peak area (%RSD below 3.73% from the instrument software and 5.87% for the Gaussian method). Combining areas reported by the high‐performance liquid chromatographic control software displayed superior limits of detection, in the order of 1 × 10^?6 M, almost an order of magnitude lower than the Gaussian method for some analytes. The introduction of the countergradient eliminated the strong solvent mismatch between dimensions, leading to a much improved peak shape and better detection limits for quantification.     

Preparative separation of isoquinoline alkaloids from Corydalis impatiens using a middle‐pressure chromatogram isolated gel column coupled with two‐dimensional liquid chromatography

We established a two‐dimensional strong cation exchange/reversed‐phase liquid chromatography protocol to isolate and purify isoquinoline alkaloids from Corydalis impatiens. Isoquinoline alkaloids were first enriched from a C. impatiens extract in which liposoluble components were removed using a medium‐pressure chromatographic tower containing middle chromatogram isolated gel. A strong cation exchange column was employed to separate and obtain 30 fractions. We chose fractions 22–29 for reversed‐phase liquid chromatography purification using characteristic isoquinoline alkaloid ultraviolet absorption spectra. Several isoquinoline alkaloid fractions (22–29) were further separated, and those of low resolution were isolated via two‐dimensional liquid chromatography in the orthogonal plane. A total of eight novel isoquinoline alkaloids with characteristic ultraviolet spectra were obtained from C. impatiens. We thus demonstrate the benefits of off‐line two‐dimensional strong cation exchange/reversed‐phase liquid chromatography to isolate isoquinoline alkaloids from C. impatiens.     

Chemometrics comparison of gas chromatography with mass spectrometry and comprehensive two‐dimensional gas chromatography with time‐of‐flight mass spectrometry Daphnia magna metabolic profiles exposed to salinity

The performances of gas chromatography with mass spectrometry and of comprehensive two‐dimensional gas chromatography with time‐of‐flight mass spectrometry are examined through the comparison of Daphnia magna metabolic profiles. Gas chromatography with mass spectrometry and comprehensive two‐dimensional gas chromatography with mass spectrometry were used to compare the concentration changes of metabolites under saline conditions. In this regard, a chemometric strategy based on wavelet compression and multivariate curve resolution–alternating least squares is used to compare the performances of gas chromatography with mass spectrometry and comprehensive two‐dimensional gas chromatography with time‐of‐flight mass spectrometry for the untargeted metabolic profiling of Daphnia magna in control and salinity‐exposed samples. Examination of the results confirmed the outperformance of comprehensive two‐dimensional gas chromatography with time‐of‐flight mass spectrometry over gas chromatography with mass spectrometry for the detection of metabolites in D. magna samples. The peak areas of multivariate curve resolution–alternating least squares resolved elution profiles in every sample analyzed by comprehensive two‐dimensional gas chromatography with time‐of‐flight mass spectrometry were arranged in a new data matrix that was then modeled by partial least squares discriminant analysis. The control and salt‐exposed daphnids samples were discriminated and the most relevant metabolites were estimated using variable importance in projection and selectivity ratio values. Salinity de‐regulated 18 metabolites from metabolic pathways involved in protein translation, transmembrane cell transport, carbon metabolism, secondary metabolism, glycolysis, and osmoregulation.     

Efficient separation of high‐purity compounds from Oxytropis falcata using two‐dimensional preparative chromatography

The separation of high‐purity compounds from traditional Tibetan medicines plays an important role in investigating their bioactivity. Nevertheless, it is often quite difficult to isolate compounds with high purity because of the complexity of traditional Tibetan medicines. In this work, an offline two‐dimensional reversed‐phase preparative method was successfully developed for the separation of high‐purity compounds from Oxytropis falcata . Based on the analysis results, an ODS C18 prep column was used for first‐dimensional preparation, and 14.8 g of the crude sample was separated into five fractions with a recovery of 74.6%. Then, an XAqua C18 prep column was used to isolate high‐purity compounds in the second‐dimensional preparation because its separation selectivity is different with the ODS C18 stationary phase. As a result, eight compounds in the crude sample were isolated in more than 98% purity. This is the first report of trans‐cinnamic acid ( 1 ) and trifolirhizin ( 2 ) from Oxytropis falcata . This method has the potential to be an efficient separation method of high‐purity compounds from Oxytropis falcata and it shows great promise for the separation of high‐purity compounds from complex samples.     

Purification of flavonoids from licorice using an off‐line preparative two‐dimensional normal‐phase liquid chromatography/reversed‐phase liquid chromatography method

An orthogonal (71.9%) off‐line preparative two‐dimensional normal‐phase liquid chromatography/reversed‐phase liquid chromatography method coupled with effective sample pretreatment was developed for separation and purification of flavonoids from licorice. Most of the nonflavonoids were firstly removed using a self‐made Click TE‐Cys (60 μm) solid‐phase extraction. In the first dimension, an industrial grade preparative chromatography was employed to purify the crude flavonoids. Click TE‐Cys (10 μm) was selected as the stationary phase that provided an excellent separation with high reproducibility. Ethyl acetate/ethanol was selected as the mobile phase owing to their excellent solubility for flavonoids. Flavonoids co‐eluted in the first dimension were selected for further purification using reversed‐phase liquid chromatography. Multiple compounds could be isolated from one normal‐phase fraction and some compounds with bad resolution in one‐dimensional liquid chromatography could be prepared in this two‐dimensional system owing to the orthogonal separation. Moreover, this two‐dimensional liquid chromatography method was beneficial for the preparation of relatively trace flavonoid compounds, which were enriched in the first dimension and further purified in the second dimension. Totally, 24 flavonoid compounds with high purity were obtained. The results demonstrated that the off‐line two‐dimensional liquid chromatography method was effective for the preparative separation and purification of flavonoids from licorice.     

Characterization of crude oil biomarkers using comprehensive two‐dimensional gas chromatography coupled to tandem mass spectrometry

Oil samples from Recôncavo basin (NE Brazil), previously analyzed by traditional techniques such as gas chromatography coupled to tandem mass spectrometry, were evaluated using comprehensive two‐dimensional gas chromatography coupled to quadrupole mass spectrometry and comprehensive two‐dimensional gas chromatography coupled to tandem mass spectrometry along with simplified methods of samples preparation to evaluate the differences and advantages of these analytical techniques to better understand the development of the organic matter in this basin without altering the normal distribution of the compounds in the samples. As a result, the geochemical parameters calculated by comprehensive two‐dimensional gas chromatography coupled to tandem mass spectrometry described better the origin, maturity, and biodegradation of both samples probably by increased selectivity, resolution, and sensitivity inherent of the multidimensional technique. Additionally, the detection of the compounds such as, the C(14α‐) homo‐26‐nor‐17α‐hopane series, diamoretanes, nor‐spergulanes, C₁₉–C₂₆ A‐nor‐steranes and 4α‐methylsteranes resolved and detected by comprehensive two‐dimensional gas chromatography coupled to tandem mass spectrometry were key to classify and differentiate these lacustrine samples according to their maturity and deposition conditions.     

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.     

Quantitative method for analysis of tobacco‐specific N‐nitrosamines in mainstream cigarette smoke by using heart‐cutting two‐dimensional liquid chromatography with tandem mass spectrometry

A heart‐cutting two dimensional liquid chromatography coupled with tandem mass spectrometry method was developed for the analysis of tobacco‐specific N‐nitrosamines (TSNAs) at low concentration level in Virginia‐type cigarette smoke. A strong cation exchange column was utilized for the first dimensional separation, which effectively removed acidic and neutral components in the smoke, followed by a reversed phase liquid chromatography coupled with tandem mass spectrometric analysis. To capture components of the TSNAs in the effluent on the trapping column, a compensating pump was applied for online dilution and pH adjustment during the period of the TSNAs fraction transferring and enrichment. Highly sensitive determination of the TSNAs in mainstream cigarette smoke was achieved by isotope deuterated internal standards under the multiple reaction monitoring mode. Compared with traditional methodologies, the method was almost no matrix interference. Limits of quantity for the TSNAs were within 0.027–0.094 ng/mL, and the results showed good reproducibility and accuracy. Finally, the new method was applied for analysis of the Kentucky reference cigarettes and the results agreed well with joint experiments of Cooperation Centre for Scientific Research Relative to Tobacco.     

Separation of phenol‐containing pyrolysis products using comprehensive two‐dimensional chromatography with columns based on pyridinium ionic liquids

We describe the application of columns with highly polar stationary liquid phases based on pyridinium ionic liquids for the two‐dimensional chromatography separation of bio‐oil and product of coal pyrolysis. By using inverse combination columns—a first ionic liquid column and a second nonpolar column—good separation results have been obtained. In the analysis of coal pyrolysis products, the suggested approach provides a much better resolution between components in comparison with a less polar first‐dimension column (based on polyethylene glycol). A good selectivity for the peaks of phenols is observed, and the group of phenols is well detached and separated from the group of diaromatics. A good separation picture was obtained also for bio‐oil, the groups of phenols and guaiacol derivatives are distinguished with good resolution of substances within each group.