Two-dimensional Liquid Chromatography Research Articles

Identification strategy of wild and cultivated Astragali Radix based on REIMS combined with two-dimensional LC-MS

A rapid and real-time method was established based on the combination of rapid evaporative ionization mass spectrometry (REIMS) and two-dimensional liquid chromatography mass spectrometry (2DLC-MS) for identification of wild Astragali Radix (WAR) and cultivated AR (CAR). The samples were analyzed under ambient ionization without time-consuming sample preparation. The phenotypic data of WAR and CAR were used to develop a real-time recognition model. Subsequently, the compounds in these two species were comprehensively characterized based on 2DLC-MS, and 45 different compounds were screened out by multivariate statistical analysis. A semi-quantitative method for 45 different compounds was established based on ultrahigh-performance liquid chromatography/quadrupole-linear ion trap mass spectrometry (UHPLC-QTRAP-MS). The results showed that the relative content of most compounds in WAR was higher than in CAR. In summary, the method has demonstrated remarkable performance in distinguishing between WAR and CAR, providing a reference in the field of traditional Chinese medicine (TCM) analysis and identification.

Harnessing the resolution power of two-dimensional liquid chromatography (2D-LC) for the screening of bisphenol contaminants in canned food items

Overlapping peaks can be difficult to avoid in 1D-LC, which make the identification and quantification of compounds ambiguous, especially if the only available detector is a UV/DAD. To overcome this, a two-dimensional liquid chromatography (2D-LC) method to monitor Bisphenols (BPs), and particularly the chosen analogues BPA, BPB, BPF, BPS, BPZ, and BPAF, in a complex matrix (canned food) was developed and validated. BPs are endocrine disruptors present in the lining of the can, which may leach into the content of canned food. It is essential to continuously identify and monitor bisphenols that are unintentionally ingested using newly available and highly efficient methods such as 2D-LC. The coefficient of variation (CV) of peak areas and retention times for all analytes in both dimensions ranged from 0.050 to 2.960 %. The recovery of the whole procedure was determined to be 67.4 ± 0.1 % in the first dimension, and 64.3 ± 0.1 % in the second dimension. The limit of detection (LOD) determined in the second dimension for the analytes ranged from 0.075 to 0.301 ppm. Finally, four canned food samples were prepared by solid-liquid extraction, and surveyed for BPA, BPB, BPF, BPS, BPZ, and BPAF. BPF (0.093 ppm) and BPAF (2.656 ppm) were accurately detected in two separate samples. This report highlights the first successful application of 2D-LC to bisphenol compounds, an important class of emerging contaminants.

Comprehensive characterization of active components in Salvia miltiorrhiza using polarity-partitioned two-dimensional liquid chromatography coupled with mass spectrometry

Salvia miltiorrhiza, a widely used traditional Chinese medicine, contains a complex matrix of hydrophobic diterpenoids and hydrophilic phenolic acids, presenting significant challenges in comprehensive analysis. In this study, an online polarity-partitioned two-dimensional liquid chromatography coupled with mass spectrometry (2D-LC-MS) method was developed for comprehensive analysis of both lipophilic and hydrophilic active components in Salvia miltiorrhiza. The method integrated hydrophilic interaction liquid chromatography (HILIC) and reversed-phase liquid chromatography (RPLC), facilitating the efficient separation of compounds across a wide range of polarities. An online dilution strategy was implemented, minimizing sample loss and enhancing the method's utility for quality control and chemical characterization of complex herbal matrices. Compared with other LC methods, this approach significantly improved analyte coverage, resolution, and analysis efficiency. Under optimal conditions, 150 active components were successfully identified, including 33 compounds newly discovered in Salvia miltiorrhiza. Additionally, the validated online method was applied to the quantitative determination of 16 quality markers of Salvia miltiorrhiza from different sources. The results demonstrated the online method's potential as a superior alternative to existing techniques, offering broader applicability in traditional Chinese medicine research.

Two-Dimensional Liquid Chromatography Tandem Mass Spectrometry Untangles the Deep Metabolome of Marine Dissolved Organic Matter.

Dissolved organic matter (DOM) is an ultracomplex mixture that plays a central role in global biogeochemical cycles. Despite its importance, DOM remains poorly understood at the molecular level. Over the last decades, significant efforts have been made to decipher the chemical composition of DOM by high-resolution mass spectrometry (HR-MS) and liquid chromatography (LC) coupled with tandem mass spectrometry (MS/MS). Yet, the complexity and high degree of nonresolved isomers still hamper the full structural analysis of DOM. To address this challenge, we developed an offline two-dimensional (2D) LC approach using two reversed-phase dimensions with orthogonal pH levels, followed by MS/MS data acquisition and molecular networking. 2D-LC-MS/MS reduced the complexity of DOM, enhancing the quality of MS/MS spectra and increasing spectral annotation rates. Applying our approach to analyze coastal-surface DOM from Southern California (USA) and open-ocean DOM from the central North Pacific (Hawaii), we annotated in total more than 600 structures via MS/MS spectrum matching, which was up to 90% more than that in iterative 1D LC-MS/MS analysis with the same total run time. Our data offer unprecedented insights into the molecular composition of marine DOM and highlight the potential of 2D-LC-MS/MS approaches to decipher the chemical composition of ultracomplex samples.

Offline Coupling of Hydrophobic Interaction Chromatography-Capillary Zone Electrophoresis for Monitoring Charge-Based Heterogeneity of Recombinant Monoclonal Antibodies.

A holistic understanding of the charge heterogeneity in monoclonal antibodies (mAbs) is paramount for ensuring acceptable product quality. Hence, biotherapeutic manufacturers are expected to thoroughly characterize their products via advanced analytical techniques. Recently, two-dimensional liquid chromatography (2DLC) methods have gained popularity for resolving complex charged species. Capillary electrophoresis (CE) is regarded as a sensitive and faster tool for charged species estimation in biotherapeutics. In this study, we aim to combine the separation power of chromatographic and electrophoretic tools (liquid chromatography [LC]-CE) so as to achieve maximum resolution of mAb charge variants. Hydrophobic interaction chromatography (HIC) has been used as the preferred LC mode with CE for achieving successful separation of both charge and hydrophobic variants for two of the mAbs (trastuzumab and rituximab). The standalone HIC and capillary zone electrophoresis (CZE) methods separated 4 hydrophobic variants and 7 charge variants for each mAb, whereas the 2DLC method separated 10 and 11 variants for mAbs A and B. On the other hand, the HIC-CZE-UV method resolved 29 variants in mAb A and 23 variants in mAb B. The reproducibility of the HIC-CZE-UV method was demonstrated by % change in values of retention time (RT) and peak area as <5% (mAb A), <3% (mAb B), and <12% (for both mAbs), respectively. Thus, the utility of the proposed LC-CE method for characterization of mAb charge variants has been displayed.

Maximizing sensitivity and selectivity in LC × LC-HRMS for pesticide analysis via exploitation of per-aqueous liquid chromatography

Accurate monitoring of pesticide residues at minimal concentrations is imperative for adherence to stringent regulatory standards in numerous countries. This study presents an innovative methodology employing comprehensive two-dimensional liquid chromatography coupled with high-resolution mass spectrometry (LC × LC-HRMS). The approach ensures high sensitivity and selectivity in detecting targeted compounds. A pivotal component of this methodology is the utilization of per-aqueous liquid chromatography (PALC) as the first dimension, facilitating the use of water-based mobile phases and addressing solvent mismatch issues. The second dimension employs reversed-phase liquid chromatography (RPLC), enhancing the separation of compounds. PALC proves instrumental in refocusing and enables the practical application of narrow-diameter columns (1.5 mm I.D.). This column design permits a direct split-free connection of the LC × LC to an electrospray-based mass spectrometer (ESI-MS), contributing to heightened sensitivity. The MS acquisition is performed in a targeted single-ion monitoring mode, ensuring reliable quantification and identification of the pesticide compounds. A comprehensive evaluation of key performance metrics, including signal-to-noise ratio, limit of detection, and response linearity, is conducted. The methodology achieves a limit of detection below the ng mL−1 and exhibits response linearity within the concentration range of 1–100 ng mL−1. The robustness of the approach is further demonstrated through intra-day and inter-day repeatability validations. Furthermore, the platform is finally tested on a surface water sample. This study not only introduces an advanced analytical methodology for pesticide multi-residue analysis but also underscores the significance of PALC in enhancing sensitivity by facilitating the use of smaller-diameter columns and water-based mobile phases, along with the role of RPLC in enhancing separation. The proposed approach showcases promising results in achieving detection limits that match the stringent regulatory standards and reliable quantification for effective pesticide residue monitoring.

Simultaneous Determination of Methotrexate Concentrations in Human Plasma and Cerebrospinal Fluid Using Two-Dimensional Liquid Chromatography: Applications in Primary Central Nervous System Lymphoma.

In this study, we aimed to develop a method for the simultaneous quantification of methotrexate (MTX) samples extracted from human plasma and cerebrospinal fluid (CSF), using two-dimensional liquid chromatography (2D-LC). Furthermore, we intended to verify whether intravenous mannitol could increase MTX concentration in the CSF of patients. The mobile phase of PUMP1 consisted of 10.0 mmol/L ammonium acetate and acetonitrile. PUMP2 solution consisted of an aqueous solution of 10.0 mmol/L ammonium acetate. The mobile phase of PUMP3 comprised 50.0 mmol/L ammonium acetate and acetonitrile, with a flow rate of 1.0 mL/min. The developed method was successfully employed to simultaneously determine drug levels in plasma and CSF from the patients treated with MTX. CSF samples were obtained by lumbar puncture 0.5 - 2 h after starting the high-dose methotrexate (HD-MTX) infusion (over 4 h) and immediately before the intrathecal (IT) administration of MTX. Venous blood samples were drawn 4 h after the start of infusion. The calibration curve was linear, with a range of 0.07 - 2.38 µmol/L for CSF samples and a range of 0.11 - 5.51 µmol/L for plasma samples. Precision (> 95%) and accuracy (> 97%) were within the acceptance criteria for each quality control (QC) level. Inter- and intra-day accuracy and precision values met the acceptance criteria for each QC level. The correlation between MTX concentrations in the plasma and CSF was moderate (r = 0.502). No significant difference was observed in MTX concentration in CSF between patients using intravenous mannitol and those not using intravenous mannitol (P = 0.682). The developed method was useful for therapeutic drug monitoring of MTX and suitable for assessing the risks and benefits of chemotherapy in patients with primary central nervous system lymphoma. Intravenous mannitol did not increase MTX concentration in the CSF of patients.

Complementarity of two-dimensional gas chromatography and two-dimensional liquid chromatography for the analysis of depolymerised lignin

Two-dimensional gas chromatography (GC × GC) and two-dimensional liquid chromatography (LC × LC) are nowadays widely used in academia and industry due to their high separation power. However, as far as we know, the complementarity of these two techniques has not yet been thoroughly studied based on the analysis of the same sample. Therefore, this was undertaken here by analysing the liquid fraction obtained after depolymerising a natural waste – lignin – with GC × GC and off-line comprehensive LC × SFC (SFC: supercritical fluid chromatography). Using complementary techniques is also important for lignin valorisation, as thorough structural characterisation of the depolymerised product can aid with developing and improving valorisation processes. For the tentative identification, NIST library was used for GC × GC–MS results and MS-DIAL together with SIRIUS for LC × SFC-MS/MS data. This allowed to study which compounds are detectable with the different 2D methods but also to discuss the limitations of the data analysis processes. The previous knowledge that LC techniques are more suitable than GC × GC for the analysis of larger oligomers and other low volatility compounds was confirmed; however, it was seen that GC × GC enabled the analysis of smaller compounds, such as aliphatic alcohols and saturated compounds. Overall, the study demonstrates the complementarity of the two techniques but also draws attention to the different detectable compound groups and classifications that the two techniques can provide.

Optimized analysis for related substances in spiramycin based on high performance liquid chromatography with hybrid particle column and characterization of its impurities by single heartcut two-dimensional liquid chromatography coupled with quadrupole time-of-flight mass spectrometer

This article described the development and validation of a method for spiramycin related substances based on hybrid particle column. The chromatographic conditions were as follows: water - 0.2 mol/L dipotassium hydrogen phosphate (the pH value adjusted to 9.5 using a 1 mol/L KOH solution) - acetonitrile - methanol (10: 60: 28.5: 1.5, v/v/v/v) as mobile phase A, water - 0.2 mol/L dipotassium hydrogen phosphate (pH 9.5) - acetonitrile - methanol (10: 30: 57: 3, v/v/v/v) as mobile phase B and gradient elution was performed. Compared with previous analytical methods, this method has strong specificity, excellent sensitivity and stability, which could be used for the daily testing of related substances of spiramycin. Furthermore, impurities above 0.1 % were characterized using two-dimensional liquid chromatography coupled with quadrupole time-of-flight mass spectrometer (2D LC-QTOF-MS/MS) and there were 6 impurities reported for the first time.

A Proteomic Study on Seed Germination of Nitraria roborowskii Kom.

Owing to the dormancy of the seeds of Nitraria roborowskii Kom., which results in a low germination rate in nature, germination takes a long time, and natural regeneration is difficult. Therefore, there is a need to study the molecular mechanism by which the seeds of N. roborowskii release dormancy. In this study, the differentially expressed proteins of N. roborowskii seeds before and after dormancy and germination were quantitatively and qualitatively analyzed via two-dimensional high-performance liquid chromatography tandem mass spectrometry (LC–MS) and TMTTM. Differentially expressed proteins from dormant and germinated seeds were characterized and enriched via bioinformatics to determine the functions and pathways of the differentially expressed proteins. The results revealed that seed dormancy was regulated by multiple metabolic pathways, including protein synthesis, nutrient utilization and phytohormone signal transduction pathways. A comparison of the dormant and germinated N. roborowskii seed samples revealed 1082 differentially expressed proteins with FC ≥ 1.5 and p values ≤ 0.05, among which 191 proteins were upregulated and 891 proteins were downregulated in the seeds of the germinated group, and proteins more closely related to the key genes of the germinated N. roborowskii seeds were involved in the activity of D-threo-aldose 1-dehydrogenase. Four proteins (WD40, cystatin, AMP binding protein, and helicase) were involved in the positive regulation of seed germination. The release of N. roborowskii seed dormancy is a complex biological process involving cell differentiation, formation, cellular transport, signaling and resistance, etc. The interactions of multiple metabolic pathways, such as carbon fixation, glycolysis/gluconeogenesis, the pentose phosphate pathway, endoplasmic reticulum protein processing and pyruvic acid metabolism in photosynthetic organisms, constitute a complex regulatory mechanism for dormancy release.

LCxSFC Valve Technologies: Guidelines toward a Successful Online Modulation.

Online two-dimensional liquid chromatography can be used under various separation modes but is sometimes limited in terms of orthogonality, especially for the analysis of neutral compounds. The use of SFC in the second dimension offers a wide choice of mobile and stationary phases, suggesting retention properties complementary to those of the first dimension. Initial works on online LCxSFC coupling published in the literature highlighted the first difficulties of solvent compatibility or potential problems with bubbles created by CO2 in loops. The present work highlights the impact of the interface between the LC and SFC dimensions on the performance of the 2D separation. Six different configurations have been evaluated, differing by the addition of a makeup flow in the first dimension, a division, or a separation of flow paths in the second dimension. Their performances with empty loops eliminated the need for complex trapping columns, regardless of the LC mobile phase nature. Injection in partial fill mode was possible without any problems of CO2 bubble formation, solvent miscibility, pressure, or modulation repeatability in each of the studied configurations. Selected configurations even allowed for the use of a mobile phase split upstream of the valve, so that the online LCxSFC configuration could be as flexible as LCxLC instrumentation in terms of the first dimension flow rate or second dimension injected volume. The obtained results allowed for building guidelines presenting the best interfaces to set up depending on the nature of the mobile phases in both dimensions. Two applications on monomers of lignin and industrial polymers confirmed preliminary results on configuration testing and illustrated that online LCxSFC could now be easily implemented. Precision tests on retention time were conclusive and are promising for the future use of this technique in industrial routines.

Abstract P100: TRPM7 deficiency amplifies aldosterone signalling cardiac fibroblasts

Introduction: Transient Receptor Potential Melastatin 7 (TRPM7) channel is a channel bound to a kinase domain that influences cellular Mg 2+ with an important role in inflammatory response and cell metabolism. TRPM7 is regulated by aldosterone. Here, we investigated the importance of TRPM7-Mg2+ in pro-fibrotic responses and oxidative stress in cardiac fibroblasts (CF) in the context of hypertension and organ damage induced by aldosterone. Methods: Wild-type (WT) and TRPM7-deficient (M7+/Δ) mice were treated with aldosterone (600µg/Kg/day) plus NaCl (1% drinking water), 4 weeks. Mechanisms were investigated in primary culture of CF. Ca 2+ and Mg 2+ influx was assessed by fluorescence microscopy and flow cytometry. Gene and protein expression was assessed by real-time PCR and immunoblotting. Proteomic analysis was performed in two-dimensional liquid chromatography and mass spectrometry (LC/LC-MS/MS). Results: M7+/Δ mice exhibited reduced tissue [Mg 2+ ] (28%) and increased cardiac collagen deposition (2-fold). Aldosterone-induced fibrosis and hypertension were enhanced in M7+/Δ. CF M7+/Δ had reduced aldosterone-induced calcium influx (136±6 vs WT:166±7) and Mg 2+ influx (1.05±0.05 vs WT 1.14±0.02). CF M7+/Δ exhibit increased expression of calpain-1(300%), α-SMA(80%), collagen-1 (50%), mineralocorticoid receptor target proteins ENaC(70%) and SGK1(30%) and reduced proliferation(33%) vs WT(p&lt;0.05). Aldosterone reduced TRPM7 (44%) and increased expression of ENaC (150%) and collagen-1 (93%) in CF WT to similar levels observed in CF M7+/Δ. CF M7+/Δ exhibit increased Nox2(70%), Nox4(120%) and ROS production (1,362 vs WT: 899 RLU/mg protein). Increased mRNA for the antioxidant catalase, NQO1 and Prdx1 (1.7-2.6-fold) was found in M7+/Δ CF. Mg 2+ supplementation normalised Ca 2+ and Mg 2+ influx, proliferation and expression of SGK1, Nox4, catalase and NQO1, whereas other effects might be mediated by the kinase domain. Proteomic analysis showed that CF M7+/Δ exhibit reduced expression of proteins related to cell migration and proliferation (Tnc, Cdcc80, Crip1, Ggt5) cell cycle and differentiation (AnxA8, Rpl22, Prickle2) and cell metabolism (Marcks, Mtab, Ces1d, Serpina3n, Afap1l1). Conclusion: Our findings demonstrate that in aldosterone-treated mice TRPM7 expression and tissue Mg 2+ levels are decreased. These processes are associated with cardiac fibrosis and oxidative stress. Our findings suggest a cardio-protective role for TRPM7/Mg2+ in aldosterone-induced hypertension.

Two-Dimensional Liquid Chromatography Purified GC/C/IRMS Doping Control Method: Analysis of Endogenous and Exogenous Sources in Urine Samples from Asian Subjects Administered a Low Dose of AICAR

AICAR (5-amino-4-imidazolecarboxyamide ribonucleoside), as a metabolic modulator, is classified in the S4 category by the World Anti-Doping Agency (WADA). Carbon Isotope Ratio Mass Spectrometry (CIR) is the mainstream method for distinguishing the endogenous and exogenous sources of AICAR in urine due to the significant individual difference in the concentration. The purpose of this study is to establish a gas chromatography combustion Isotope Ratio Mass Spectrometry (GC/C/IRMS) method for AICAR based on efficient two-dimensional liquid chromatography (2D-HPLC) separation. MethodIn this study, an automated 2D-HPLC separation technique was used to separate and purify AICAR and endogenous reference substances in urine samples. Then, AICAR was derivatized with 3-TMS as the main derivative product, while the endogenous reference compounds remained in their original form. Subsequently, the developed GC/C/IRMS method was utilized for the detection of the target and reference substances. Followed, we evaluated the applicability of this method using urine samples from two Asian males administered a low dose of AICAR (3 grams). ResultsThe advantages of this study include: 1) reduced sample pretreatment time: the established 2D-HPLC separation method can separate the target and endogenous reference substances in one step; 2) low interference: the isotope chromatograms have low background interference, and the separation of endogenous reference substances is purer; 3) more accurate result calculations: this method only requires derivatization and result correction for AICAR, with the endogenous reference substances measured in their original form, reducing biases from corrections of multiple substances. The detection method performed well, with a concentriton limit of 2500 ng/mL, meeting the needs of routine detection concentrations. The CIR results from volunteer samples indicated that samples collected within 16 hours post-administration exceeded the threshold set in the literature. ConclusionThis study successfully established a 2D-HPLC-GC/IRMS method that integrates CIR as the most stable indicator for distinguishing the internal and external sources of AICAR. After administering a low dose of AICAR to the Asian population, exogenous drug characteristics were manifested within 16 hours. This observation, when compared to the 40-hour detection window cited in the literature, suggests that the length of the detection window is positively correlated with the dosage of the test drug.

Consolidating two-dimensional liquid chromatography–high-resolution tandem mass spectrometry (LC×LC-HRMS/MS) technique for the non-targeted analysis of poly- and perfluorinated substances: A trial on aqueous film-forming foams

To date, poly- and perfluoroalkyl substances (PFAS) represent a real threat for their environmental persistence, wide physicochemical variability, and their potential toxicity. Thus far a large portion of these chemicals remain structurally unknown. These chemicals, therefore, require the implementation of complex non-targeted analysis workflows using liquid chromatography coupled with high-resolution mass spectrometry (LC-HRMS) for their comprehensive detection and monitoring. This approach, even though comprehensive, does not always provide the much-needed analytical resolution for the analysis of complex PFAS mixtures such as fire-fighting aqueous film-forming foams (AFFFs). This study consolidates the advantages of the LC×LC technique hyphenated with high-resolution tandem mass spectrometry (HRMS/MS) for the identification of PFAS in AFFF mixtures. A total of 57 PFAS homolog series (HS) were identified in 3M and Orchidee AFFF mixtures thanks to the (i) high chromatographic peak capacity (n’2D,c ~ 300) and the (i) increased mass domain resolution provided by the “remainder of Kendrick Mass” (RKM) analysis on the HRMS data. Then, we attempted to annotate the PFAS of each HS by exploiting the available reference standards and the FluoroMatch workflow in combination with the RKM defect by different fluorine repeating units, such as CF2, CF2O, and C2F4O. This approach resulted in 12 identified PFAS HS, including compounds belonging to the HS of perfluoroalkyl carboxylic acids (PFACAs), perfluoroalkyl sulfonic acids (PFASAs), (N-pentafluoro(5)sulfide)-perfluoroalkane sulfonates (SF5-PFASAs), N-sulfopropyldimethylammoniopropyl perfluoroalkane sulfonamides (N-SPAmP-FASA), and N-carboxymethyldimethylammoniopropyl perfluoroalkane sulfonamide (N-CMAmP-FASA). The annotated categories of perfluoroalkyl aldehydes and chlorinated PFASAs represent the first record of PFAS HS in the investigated AFFF samples.

Artificial intelligence decision making tools in food metabolomics: Data fusion unravels synergies within the hazelnut (Corylus avellana L.) metabolome and improves quality prediction

This study investigates the metabolome of high-quality hazelnuts (Corylus avellana L.) by applying untargeted and targeted metabolome profiling techniques to predict industrial quality. Utilizing comprehensive two-dimensional gas chromatography and liquid chromatography coupled with high-resolution mass spectrometry, the research characterizes the non-volatile (primary and specialized metabolites) and volatile metabolomes. Data fusion techniques, including low-level (LLDF) and mid-level (MLDF), are applied to enhance classification performance. Principal component analysis (PCA) and partial least squares discriminant analysis (PLS-DA) reveal that geographical origin and postharvest practices significantly impact the specialized metabolome, while storage conditions and duration influence the volatilome. The study demonstrates that MLDF approaches, particularly supervised MLDF, outperform single-fraction analyses in predictive accuracy. Key findings include the identification of metabolites patterns causally correlated to hazelnut’s quality attributes, of them aldehydes, alcohols, terpenes, and phenolic compounds as most informative. The integration of multiple analytical platforms and data fusion methods shows promise in refining quality assessments and optimizing storage and processing conditions for the food industry.

Determination of pesticide residues in five different corn-based products using a single and simple solid–liquid extraction without clean-up steps followed by comprehensive two-dimensional liquid chromatography coupled to tandem mass spectrometry

Pesticide residues in food products are a critical issue concerning food safety. Recent studies have increasingly focused on developing selective, rapid, innovative, and environmentally friendly analytical methods for detecting pesticides in foodstuffs. This study presents a novel analytical method utilizing comprehensive two-dimensional liquid chromatography-tandem mass spectrometry for the simultaneous determination of one hundred and thirteen multiclass pesticides in corn products. The method simplifies the process with a single-step solid–liquid extraction and benefits from two-dimensional separation to enhance resolution and minimize matrix effects. The methodology was validated according to European Commission DG-SANTE (SANTE/11312/2021) and European Committee for Standardization (EN 15662, 2018) guidelines, obtaining acceptable analytical parameters. Limits of detection ranged from 0.2 µg/L to 30.2 µg/L and limits of quantification from 0.3 to 91.6 μg/L, all bellow regulatory limits. Accuracy, ranged from 60 to 120 % and precision, estimated by relative standard deviation, was consistently below 15.1 % in all cases. The applicability of the method was demonstrated by analyzing pesticides in five representative corn products (tortilla, corn flakes, corn cake, starch, and polenta). Sixteen pesticides were detected, all within the regulatory residue limits. Three metric tools were applied to assess the analytical process. Environmental sustainability was evaluated using the AGREE metric, while analytical performance and method applicability were assessed with the Red-Green-Blue and Blue Applicability Grade Index models, respectively, yielding promising scores (0.5, ≥ 0.55, and 0.65). These findings and the comparison to previous methods indicated that this approach represents a promising, alternative and user-friendly strategy for pesticide determination, offering effective productivity and reliable analytical performance.

Development of a Two-Dimensional Liquid Chromatography Online Deproteinization Method for Determining Paclitaxel-Related Substances in a Paclitaxel Injection (Albumin-Bound)

To determine the paclitaxel-related substances in a paclitaxel injection (albumin-bound), it is necessary to remove human blood albumin from the sample via solid-phase extraction and subsequently perform high-performance liquid chromatography analysis. The pre-processing operation is complicated, the duration of analysis is long, and much human power and material resources are needed in the sample testing process. Thus, the purpose of this study was to develop a quick two-dimensional liquid chromatography method that requires less consumables and possesses high sensitivity. Samples were directly injected into one-dimensional solid-phase extraction columns to remove human blood albumin. Paclitaxel and its related substances in the solid-phase extraction columns were then eluted into two-dimensional analytical columns through valve switching technology. The sample pretreatment step can be omitted in this method, which allows for the analysis of paclitaxel and its related substances to be completed more quickly, within only 15 min, under the developed two-dimensional elution conditions. The established method had good linearity (R2 = 0.9994), limit of detection (0.25 μg·mL−1), limit of quantification (0.5 μg·mL−1), precision (RSD = 0.5%), and accuracy (102.53–107.97%).

Analysis of Antioxidant Compounds in Vitex negundo Leaves Using Offline 2D-LC-ECD and LC-MS/MS.

Vitex negundo has strong antioxidant activity, but its primary antioxidant components are not clear. In this study, the antioxidant components were screened by offline two-dimensional liquid chromatography coupled with electrochemical detection (2D-LC-ECD) and subsequently assessed using liquid chromatography-tandem mass spectrometry (LC-MS/MS) identification, radical scavenging capacity, and molecular docking. Various fractions were isolated from Vitex negundo leaves, and 39 antioxidant components were screened and identified. All of the fractions containing the antioxidant components exhibited certain antioxidant activity. Correlation analysis revealed a strong correlation between the response of LC-ECD and the in vitro antioxidant activity of the fractions. Molecular docking demonstrated that components with high response to LC-ECD exhibited robust interaction with antioxidant-related target proteins. The main antioxidant components of Vitex negundo leaves were isoorientin, chlorogenic acid, agnuside, cynaroside, and scutellarin. The 2D-LC-ECD combined with LC-MS/MS was rapid and effective in screening the antioxidant components in Vitex negundo leaves and could also provide technical support for the discovery of antioxidant components with different polarities and contents in other medicinal and edible plants.

Non-enantioselective, enantioselective, and two-dimensional liquid chromatography coupled with tandem mass spectrometry for the study of stereochemical disposition of oxylipins in cGMP-regulated hemin-treated platelets

Oxylipins are important low abundant signaling molecules in living organisms. In platelets they play a primary role in platelet activation and aggregation in the course of thrombotic events. In vivo, they are enzymatically synthesized by cyclooxygenases, lipoxygenases, or cytochrome P450 isoenzmes, resulting in diverse polyunsaturated fatty acid (FA) metabolites including hydroxy-, epoxy-, oxo-FAs, and endoperoxides with pro-thrombotic or anti-thrombotic effects. In a recent study, it was reported that hemin induces platelet death which was accompanied by enhanced reactive oxygen species (ROS) production (measured by flow cytometry) and lipid peroxidation (as determined by proxy using flow cytometry with BODIPY-C11 as sensor). Lipidomic studies further indicated significant changes of the platelet lipidome upon ex vivo hemin treatment, amongst others oxylipins were increased. The effect could be (at least partly) reversed by riociguat/diethylamine NONOate diethylammonium salt (DEA/NO) which modulates the soluble guanylate cyclase(sGC)-cGMP-cGMP-dependent protein kinase I(cGKI) signaling axis. In the original work, oxylipins were measured by a non-enantioselective UHPLC-tandem-MS assay which may not give the full picture whether oxylipin elevation is due to ROS or by enzymatic processes. We present here the study of the stereochemical disposition of hemin-induced platelet lipidome alterations using Chiralpak IA-U column with amylose tris(3,5-dimethylphenylcarbamate) chiral selector immobilized on 1.6 µm silica particles. It was found that the major platelet oxylipins 12-HETE, 12-HEPE and 14-HDoHE (from 12-LOX) and 12-HHT (from COX-1) were present in S-configuration indicating their enzymatic formation. On the other hand, both R and S enantiomers of 9- and 13-HODE, 11- and 15-HETE were detected, possibly due to enzyme promiscuity rather than non-specific oxidation (by ROS or autoxidation), as confirmed by multi-loop based two-dimensional LC-MS using selective comprehensive mode with achiral RPLC in the 1st dimension and chiral LC in the 2nd using a multiple heart-cutting interface. For 12-HETrE, a peak at the retention time of the R-enantiomer was ruled out as isobaric interference by 2D-LC-MS. In particular, arachidonic acid derivates 12(S)-HHT, 11(R)-HETE and 15(S)-HETE were found to be sensitive to hemin and cGMP modulation.

Eyes on the Prize: Overcoming Uncertainty to Realize the Power of 2D-LC Separations

Two-dimensional liquid chromatography (2D-LC) is a technique that extends the separation capabilities of conventional liquid chromatography by adding a second separation step to resolve compounds that are coeluted from a first column. This approach holds tremendous potential to solve difficult separation challenges in fields ranging from pharmaceutical analysis to biofuel characterization. Currently, method development is a significant bottleneck impeding more widespread implementation of 2D-LC methods, particularly for new users who are uncertain about how to proceed. In this month’s column, I highlight some of the primary considerations we face in method development and point to resources that can help users overcome uncertainty and develop highly effective 2D-LC methods.