Heart-cutting Two-dimensional Liquid Chromatography Research Articles

An Analysis of Polysaccharides from Eight Plants by a Novel Heart-Cutting Two-Dimensional Liquid Chromatography Method.

Natural polysaccharides are important active biomolecules. However, the analysis and structural characterization of polysaccharides are challenging tasks that often require multiple techniques and maps to reflect their structural features. This study aimed to propose a new heart-cutting two-dimensional liquid chromatography (2D-LC) method for separating and analyzing polysaccharides to explore the multidimensional information of polysaccharide structure in a single map. That is, the first-dimension liquid chromatography (1D-LC) presents molecular-weight information, and the second-dimension liquid chromatography (2D-LC) shows the fingerprints of polysaccharides. In this 2D-LC system, the size-exclusion chromatography-hydrophilic interaction chromatography (SEC-HILIC) model was established. Coupling with a charged aerosol detector (CAD) eliminated the need for the derivatization of the polysaccharide sample, allowing the whole process to be completed within 80 min. The methods were all validated in terms of precision, linearity, stability, and repeatability. The capability of the new 2D-LC method was demonstrated in determining various species of natural polysaccharides. Our experimental data demonstrated the feasibility of the whole systematic approach, opening the door for further applications in the field of natural polysaccharide analysis.

Structural Identification of Impurities in Pioglitazone Hydrochloride Preparations by 2D-UHPLC-Q-Exactive Orbitrap HRMS and Their Toxicity Prediction.

Pharmaceutical companies and regulatory agencies have more and more concerns for impurities in pharmaceuticals and their toxicity. In this work, heart-cutting two-dimensional ultrahigh-performance liquid chromatography (2D-UHPLC) in combination with high-resolution mass spectrometry (HRMS) was used, setting HRMS as positive mode of electrospray ionization to identify five impurities in pioglitazone hydrochloride preparations. With the heart-cutting 2D-UHPLC and online desalting technique, the structures of five impurities were deduced in an analysis of MSn data. And three of them, Impurity-2, Impurity-3, and Impurity-5, have never been reported before. The fragmentation patterns of five impurities were proposed on a basis of accurate mass and fragment ions in this study. Since the toxicity of impurities is relevant to their structures, toxicology of all five impurities was predicted by three software tools, and the result showed that these compounds have good safety profile.

Simultaneous separation and determination of several chiral antidepressants and their enantiomers in wastewater by online heart-cutting two-dimensional liquid chromatography

A novel method for simultaneous separation and detection of the racemates and the enantiomers of common chiral antidepressants in wastewater matrix was developed by online heart-cutting two-dimensional liquid chromatography (2D-LC) coupled to solid-phase extraction (SPE). Screening of chiral stationary phases (CSPs) and chromatographic conditions was investigated for complete enantioseparation to be compatible with RP-HPLC in 1st D-LC. Using methanol-0.1 % (v/v) ammonia solution as mobile phase, a 2D-LC system was configured by reversed mode with a combination of C18 column and the serially CPS columns as 2D-LC stationary phases respectively. The target analytes could achieve satisfactory transformation between 2D-LCs with transfer rate of 90.57–98.58 %. By means of freeze-drying and SPE, three antidepressants in wastewater were greatly preconcentrated under the optimized conditions, improving the method performance. The racemates and the enantiomers of mirtazapine, bupropion and fluoxetine exhibited good linearity in the range of 0.10–30.00 ng/mL (R2≥0.9986), and LODs and LOQs ranged in 0.0183–0.0549 ng/mL and 0.0661–0.1831 ng/mL, respectively. By this way, the method was successfully applied to simultaneous determination of the racemates and the enantiomers of mirtazapine, bupropion and fluoxetine in wastewater samples. Among them, three samples contained bupropion at level of 0.401–0.822 ng/mL, and mirtazapine at level of 0.328 and fluoxetine at level of 0.381 ng/mL were detected respectively in the other two samples. The enantiomers were at level of 0.140–0.189 ng/mL for mirtazapine, 0.182–0.419 ng/mL for bupropion and 0.179–0.204 ng/mL for fluoxetine, respectively. The proposed method providing an efficient approach to monitoring chiral drugs and their enantiomers in wastewater, facilitating to pollution assessment of chiral drugs in the environment and regional survey of illicit abuse in drug control.

An on-line heart-cutting two-dimensional liquid chromatography method for intracellular 2-hydroxyglutarate enantiomers.

D-2-Hydroxyglutarate (D-2-HG) is an oncometabolite that induces cancer cell survival and growth. D-2-HG is produced by mutations in isocitrate dehydrogenases 1 and 2. L-2-HG has different roles than the D-form, and chiral discrimination is important for elucidating the exact roles of the 2-HG enantiomers. In this study, an analytical method for 2-HG enantiomers was developed using on-line heart-cutting two-dimensional liquid chromatography (2D-LC) with fluorescence detection. Fluorescence derivatization of 2-HG with 4-nitro-7-piperazino-2,1,3-benzoxadiazole (NBD-PZ) was performed using 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride, a hydrophilic condensing reagent, at 70 °C for 30 min. The first dimension on the octadecylsilyl column was aimed at separating NBD-PZ-2-HG from other compounds obtained via derivatization or from biological fluids. The NBD-PZ-2-HG peak was fractionated into a sample loop and automatically injected into the second dimension. In the second dimension, a CHIRALPAK IC column separated NBD-PZ-D- and L-2-HG with a resolution of 2.14. The limits of quantification were 0.25 pmol per injection for NBD-PZ-D-2-HG and-L-2-HG. The precision values were below 6.58%, and the accuracies were 88.2-92.8%. The intracellular concentrations of D-2-HG and L-2-HG in the cancer cells were 13.5 ± 0.4 and 9.9 ± 0.3 pmol per 1.0 × 106 cells, respectively. The developed method will be useful for elucidating the role of 2-HG enantiomers in cancer cells.

Differentiating root and rhizome of panax notoginseng based on precursor ion scanning and multi heart-cutting two-dimensional liquid chromatography.

Owing to increasing demand for Panax notoginseng-based medicines and health products, establishing a fast, simple, and reliable assay to analyze the chemical differences between its root and rhizome is important. Although previous studies showed that the chemical and biological differences between the root and rhizome of P. notoginseng seem to be small, efforts should be taken to investigate such differences to ensure the safety and efficacy of the products. This work describes a holistic approach that combines characteristic fingerprinting using ultra-high performance liquid chromatography-tandem mass spectrometry parent ion scanning with charged aerosol detection and targeted separation by online heart-cutting two-dimensional liquid chromatography, to identify and evaluate characteristic markers allowing differentiation of the root and rhizome. A total of five potential markers chikusetsusaponin L5 , ginsenoside Rb2 , stipuleanoside R2, malonyl-ginsenoside Rb1 , and malonyl-ginsenoside Rd, were identified and confirmed by comparing chromatographic retention time, the accurate mass of molecular weight, and the fragments of secondary MS with the available reference materials. The results showed that all five markers were 2.8-7 times higher in content in the rhizome than in the root.

Application of heart-cutting two-dimensional liquid chromatography-mass spectrometry to the characterization of highly polar impurities in calcium gluconate injection

The separation and characterization of small polar impurities in polar drugs such as calcium gluconate products are always challenging, due to their poor retention on traditional reversed phase (RP) columns. Although ion-pair reversed-phase liquid chromatography (IPRP-LC) and hydrophilic interaction liquid chromatography (HILIC) are commonly used methods for polar compound analysis, both methods have some drawbacks. For example, IPRP-LC is incompatible with mass spectrometry (MS) due to the presence of non-volatile salts in its mobile phase and HILIC has limited sensitivity due to the poor solubility of polar drugs in the organic-rich sample diluents used in HILIC separations. In order to characterize the highly polar impurities in calcium gluconate injections, a heart-cutting two-dimensional liquid chromatography (2D-LC) method coupled with quadrupole time-of-flight mass spectrometry (Q-TOF/MS) was developed in this study. An IPRP-LC method in the first dimension (1D) provided the selectivity for the separation of polar analytes, using a 100% aqueous mobile phase containing phosphate buffer and ion-pair reagent. Heart cuts of target peaks were collected with sample loops and transferred to the second dimension (2D) HILIC column using an organic-rich mobile phase. In order to solve the mobile phase mismatch problem between the two dimensions, a make-up flow module was introduced in the 2D-LC system to dilute the 1D-water-rich fractions with acetonitrile before entering the sample loops. By optimizing the loop size and dilution factor, good retention and peak shape of the highly polar impurities were obtained on the 2D-HILIC column, and the ion suppression effect for MS detection from the ion-pair reagent and non-volatile salt in the 1D-effluent was minimized. A total of five impurities were identified through fragmentation studies by Q-TOF/MS analysis and their fragmentation pathways were proposed. Four of them were further confirmed by reference substances. This study not only provided useful information for quality control of calcium gluconate injections, but also provided an alternative method for polar impurity characterization in pharmaceuticals.

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.

Two-dimensional liquid chromatography - mass spectrometry as an effective tool for assessing a wide range of pharmaceuticals and biomarkers in wastewater-based epidemiology studies

The biomarkers used in wastewater-based epidemiology studies have a wide range of physicochemical properties, making simultaneous analysis extremely challenging or even impossible. So far, the majority of analytical procedures employed in this field have focused on specific classes or a limited number of analytes. The heart-cutting two-dimensional liquid chromatography tandem mass spectrometry technique (2D-LC–MS/MS) has enabled the incorporation of compounds with significantly different polarities in a single analytical method.In this study, a 2D-LC–MS/MS method has been developed and optimized for the simultaneous analysis of various biomarkers in wastewater, providing relevant information about lifestyle habits (the usage of alcohol, tobacco, and caffeine), health and well-being (pharmaceuticals), and a population size biomarker (5-HIAA) that is useful for correct calculation of human population during wastewater-based epidemiology studies. A single-laboratory validation procedure showed satisfactory analytical performance, with the mean trueness for most compounds falling in the range of 80÷110 % and the mean repeatability being less than 20 %. The applicability of the method was evaluated by testing 28 wastewater samples collected at different wastewater treatment plants in Latvia. The obtained results revealed the occurrence of 44 out of 62 selected biomarkers, with the lowest quantified concentration at 1.1 ng L−1 in the case of xylometazoline and up to 148 μg L−1 in the case of metformin and 156 μg L−1 for caffeine.

A novel and sensitive method for determination of amisulpride in human plasma by two-dimensional liquid chromatography.

A novel and sensitive heart-cutting two-dimensional liquid chromatography with ultraviolet detection method (2D-LC-UV) was developed and validated for determination of amisulpride in human plasma. The 2D-LC system consists of a first dimensional (1 D) LC column and a middle transfer column as well as a second-dimensional (2 D) LC column. After simple protein precipitation, the sample was directly injected into the introduction valve of the 2D-LC system. The 1 D column, playing a role of primary separation and preconcentration for complex plasma matrices, transferred the targets to the intermediate column. Following capture of targets on the middle column online, the analytes were transferred to the 2 D separation column by a six-port valve. The 2 D column, avoiding interference from the plasma matrix, completed further separation and quantification. An assistant pump was optimized for primary enrichment as well as final elution in the heart-cutting mode. The analytical time of amisulpride was 7.401 min. The accuracy was between 0.48 and 8.49%, while the intra- and inter-day precisions ranged from 0.9 to 3.1% and from 1.7% to 3.3%, respectively. The linear range of amisulpride was 48.15-2,407.59 ng/ml, while the extraction recovery was 98.7-101.3%. The strategy established in the study, which was successfully applied to therapeutic drug monitoring of amisulpride for routine clinical detection, displays high sensitivity, good repeatability, convenience and low cost.

Ginsenoside Contents in Ginseng: Quality by Design-Coupled Two-Dimensional Liquid Chromatography Technique

Red ginseng and white ginseng, with different chemical constituents, exhibit different antioxidative, anticancer, antiasthmatic and immunomodulatory properties. The aim of this study was to determine the amount of ginsenoside contents (Rg1, Re, Rb1, Rb2, Rc, Rd and Ro) in red and white ginseng. A rapid and comprehensive method was developed using the quality-by-design (QbD) and heart-cutting two-dimensional liquid chromatography (2D-LC) techniques. The temperature (25°C), mobile phase constituent (0.1%H3PO4), flow rate (0.35mL/min) and concentrations of the final (45%) and initial (19.5%) organic solvents were optimized to efficient chromatography-based isolation method. The gradient program was optimized by QbD Fusion AE system. A selective column (Thermo Acclaim RSLC Polar Advantage II 2.2μm, 100×2.1mm) was used for the studies. The ginsenoside Rb1, Rc and Ro exhibiting poor separation resolution were separated using the heart-cutting 2D-LC technique. The average Rb1, Rb2 and Rc contents in red ginseng were significantly higher than the average Rb1, Rb2 and Rc contents in white ginseng. Ginsenoside Ro can be potentially used as a marker to evaluate the qualities of white and red ginseng. This comprehensive and rapid method can be potentially used to screen the quality of the markers in the future.

Multiple Heart-Cutting Two-Dimensional Liquid Chromatography: Recent Developments and Applications

Background: Over the last few decades, there has been a growing interest in Two- Dimensional Liquid Chromatography (2D-LC). This advanced technology has become a relevant solution for solving all the analytical challenges encountered in the pharmaceutical industry. The primary purpose of this review is to give a brief account of the developing techniques notable in 2D-LC. Methods: Review of recent literature on advanced separation techniques for complex mixtures revealed that 2D-LC is the most widely used technique in the pharmaceutical laboratory for the separation of complex samples. Among the various techniques used in 2D-LC, comprehensive 2D-LC and heart-cutting 2D-LC are highly used techniques. To enhance the resolution, further development has been made to heart-cutting 2D-LC called multiple heart-cutting 2D-LC, which has recently become a very popular technique in the pharmaceutical industry. Therefore research articles mentioning the use of multiple heart-cutting 2D-LC for separation of complex mixtures were reviewed. Results: The most crucial benefit of multiple heart-cutting 2D-LC is its reduced effort in method development and also the storage of multiple sample sections for further analysis. The multiple heartcutting 2D-LC is suited, preferably for method development and impurity analysis of pharmaceutical substances, fine chemicals and offers better peak separation, which is otherwise not possible by comprehensive 2D-LC. Conclusion: The advantage of two-dimensional liquid chromatography over one-dimensional liquid chromatography is its high resolving power. Further improvement in resolution was achieved by using the advanced multiple heart-cutting 2D-LC which appears to have an imperative role in pharmaceutical and food analysis.

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.

Heart-cutting two-dimensional liquid chromatography coupled to quadrupole-orbitrap high resolution mass spectrometry for determination of N,N-dimethyltryptamine in rat plasma and brain; Method development and application

The orthogonal heart-cutting liquid chromatography (LC) modes coupled to high-resolution tandem mass spectrometry (HRMS/MS) provide a number of possibilities to enhance selectivity and sensitivity for the determination of targeted compounds in complex biological matricies. Here we report the development of a new fast 2D-LC-(HRMS/MS) method and its successful application for quantitative determination of the level of plasma and brain N,N-dimethyltriptamine (DMT) using α-methyltryptamine (AMT) as internal standard in an experimental model of cerebral ischemia/reperfusion using DMT administration. The 2D-LC separation was carried out by a combination of hydrophilic interaction liquid chromatography (HILIC) in the first dimension followed by second-dimensional reversed-phase (RP) chromatography within a total run time of 10 min. The enrichment of HILIC effluent of interest containing DMT was performed using a C18 trapping column. During method development several parameters of sample preparation procedures, chromatographic separation and mass spectrometric detection were optimised to achieve high DMT recovery (plasma: 90 %, brain: 88 %) and sensitivity (plasma: 0.108 ng/mL of LOD, brain: 0.212 ng/g of LOD) applying targeted analytical method with strict LC and HRMSMS confirmatory criteria.Concerning rat plasma sample, the concentration of DMT before hypoxia (49.3–114.3 ng/mL plasma) was generally higher than that after hypoxia (10.6–96.1 ng/mL plasma). After treatment, the concentration of DMT in brain was elevated up to the range of 2–6.1 ng/g.Overall, our analytical approach is suitable to detect and confirm the presence of DMT administered to experimental animals with therapeutic purpose in a reliable manner.

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.

Bias reduction in the quantitative analysis of a target analyte present in a limited quantity in human plasma using dual-mode heart-cutting two-dimensional liquid chromatography coupled with isotope dilution mass spectrometry.

Dual-mode heart-cutting two-dimensional liquid chromatography (DMHC 2D-LC) was applied to isotope dilution mass spectrometry (IDMS) to reduce the bias in the quantitative analysis of a target analyte present in a limited quantity in human plasma. Based on a Waters I-Class LC system, the DMHC 2D-LC system was operated in one- and two-dimensional modes to facilitate the determination of heart-cutting time and the efficient trapping of the target LC eluate. Experiments to determine the feasibility of coupling with IDMS were performed with triple quadrupole mass spectrometry using folic acid standards and/or 13 C5 -folic acid. To validate the performance of the DMHC 2D-LC/IDMS system on a complex sample, human plasma was analyzed for folic acid and the result was compared with that obtained using conventional single-column LC. The total run time of the DMHC 2D-LC system was 20 min, the same as that of the single-column LC system. The peak profile of the spiked 13 C5 -folic acid obtained with single-column LC/MS was affected by matrix effects, but resolved with DMHC 2D-LC/MS, thus improving the accuracy of the analysis. The DMHC 2D-LC/IDMS system showed reliable performance in analyzing the target analyte in human plasma, eliminating matrix effects and saving analysis time.

Determination of mycotoxins in beer by multi heart-cutting two-dimensional liquid chromatography tandem mass spectrometry method

Beer is one the most consumed alcoholic beverage in the world and its contamination with mycotoxins is of public health concern. This study reports a fast and automated analytical procedure based on a multi-heart-cutting two-dimensional liquid chromatography tandem mass spectrometry method using electrospray ionization for the determination of seven mycotoxins (aflatoxins B1, B2, G2 and G1, ochratoxin A, fumonisins B1 and B2) in beers. The developed method was based on the heart-cutting 2D- HPLC technique in which only the specific portions of the first dimension, in the retention time of analytes, were transferred into the second dimension for the further separation and successive determination. The method uses two different chromatographic columns; in the first dimension, 50 μL of sample was injected on first column, and mycotoxins elution regions were collected in a loop and transferred into the second column for the separation of analytes. Each column operated in gradient elution mode in order to eliminate interfering compounds and improve separation and peak shape. After the optimization, the method has been validated according to EU regulation and finally applied for the analysis of forty beer samples collected from Italian supermarkets. Among all mycotoxins studied, fumonisins B1 was the most widely distributed in analysed beers (>21%) in the range from 0.6 to 12.3 ng mL−1. The automated methodology developed was able to determine accurately and simultaneously seven mycotoxins in beer. This provided a significant reduction of sample handle and, consequently of analysis time.

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 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.

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.

Heart-cutting two-dimensional liquid chromatography combined with isotope ratio mass spectrometry for the determination of stable carbon isotope ratios of gluconic acid in honey

Liquid chromatography/isotope ratio mass spectrometry (LC/IRMS) is used to analyze various types of samples, including foodstuffs, to determine their authenticity and trace their origin on the basis of their stable carbon isotope ratios (δ13C). However, multicomponent samples are difficult to analyze. For example, determining the δ13C values of the organic acids in honey is complicated by the presence of large amounts of carbohydrates. Herein, we present a heart-cutting two-dimensional LC/IRMS method for analysis of honey samples. In this method, the organic acids in the samples were first separated from the carbohydrates by a size-exclusion column, and then the organic acids were separated from each other by a reverse-phase column connected to the first column via a switching valve. By means of this method, the δ13C values for three organic acids in high-carbohydrate-content simulated honey samples could be determined with high accuracy and precision (≤0.3‰ and ≤0.1‰, respectively). In addition, the gluconic acid δ13C values for 25 honey samples were determined with high precision and found to range from −31.7 to −28.5‰ (mean: −30.0 ± 0.7‰). These values shed some light on the mechanism of gluconic acid production. Taken together, our results suggest that this two-dimensional LC method has the potential to be more effective than one-dimensional LC for use in isotopic research.