On-line Two-dimensional Liquid Chromatography Research Articles

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

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

Development of a rapid screening method utilizing 2D LC for effect-directed analysis in the identification of environmental toxicants

Effect-directed analysis (EDA) is a crucial tool in environmental toxicology, effectively integrating toxicity testing with chemical analysis. The conventional EDA approach, however, presents challenges such as significant solvent consumption, extended analysis time, labor intensity, and potential contamination risks. In response, we introduce an innovative alternative to the conventional EDA. This method utilizes the MTT bioassay and online two-dimensional liquid chromatography (2D LC) coupled with high-resolution mass spectrometry (HR-MS), significantly reducing the fractionation steps and leveraging the enhanced sensitivity of the bioassay and automated chemical analysis. In the chemical analysis phase, a switching valve interface is employed for comprehensive analysis. We tested the performance of both the conventional and our online 2D LC-based methods using a household product. Both methods identified the same number of toxicants in the sample. Our alternative EDA is 22.5 times faster than the conventional method, fully automated, and substantially reduces solvent consumption. This novel approach offers ease, cost-effectiveness, and represents a paradigm shift in EDA methodologies. By integrating a sensitive bioassay with online 2D LC, it not only enhances efficiency but also addresses the challenges associated with traditional methods, marking a significant advancement in environmental toxicology research.

A comprehensive investigation on the chemical changes of traditional Chinese medicine with classic processing technology: Polygonum multiflorum under nine cycles of steaming and sunning as a case study.

The processing of traditional Chinese medicine (TCM) plays an important role in the clinical application, which usually has the function of "increasing efficiency and reducing toxicity". Polygonum multiflorum (PM) has been reported to induce hepatotoxicity, while it is believed that the toxicity is reduced after processing. Studies have shown that the hepatotoxicity of PM is closely related to the changes in chemical components before and after processing. However, there is no comprehensive investigation on the chemical changes of PM during the processing progress. In this research, we established a comprehensive method to profile both small molecule compounds and polysaccharides from raw and different processed PM samples. In detail, an online two-dimensional liquid chromatography coupled with quadrupole-orbitrap mass spectrometry (2D-LC/Q-Orbitrap MS) was utilized to investigate the small molecules, and a total of 150 compounds were characterized successfully. After multivariate statistical analysis, 49 differential compounds between raw and processed products were screened out. Furthermore, an accurate and comprehensive method for quantification of differential compounds in PM samples was established based on ultra-high performance liquid chromatography/Q-Orbitrap-MS (UHPLC/Q-Orbitrap-MS) within 16 min. In addition, the changes of polysaccharides in different PM samples were analyzed, and it was found that the addition of black beans and steaming times would affect the content and composition of polysaccharides in PM significantly. Our work provided a reference basis for revealing the scientific connotation of the processing technology and increasing the quality control and safety of PM.

Pioneering Just-in-Time (JIT) Strategy for Accelerating Raman Method Development and Implementation for Biologic Continuous Manufacturing.

Raman spectroscopy is a popular process analytical technology (PAT) tool that has been increasingly used to monitor and control the monoclonal antibody (mAb) manufacturing process. Although it allows the characterization of a variety of quality attributes by developing chemometric models, a large quantity of representative data is required, and hence, the model development process can be time-consuming. In recent years, the pharmaceutical industry has been expediting new drug development in order to achieve faster delivery of life-changing drugs to patients. The shortened development timelines have impacted the Raman application, as less time is allowed for data collection. To address this problem, an innovative Just-in-Time (JIT) strategy is proposed with the goal of reducing the time needed for Raman model development and ensuring its implementation. To demonstrate its capabilities, a proof-of-concept study was performed by applying the JIT strategy to a biologic continuous process for producing monoclonal antibody products. Raman spectroscopy and online two-dimensional liquid chromatography (2D-LC) were integrated as a PAT analyzer system. Raman models of antibody titer and aggregate percentage were calibrated by chemometric modeling in real-time. The models were also updated in real-time using new data collected during process monitoring. Initial Raman models with adequate performance were established using data collected from two lab-scale cell culture batches and subsequently updated using one scale-up batch. The JIT strategy is capable of accelerating Raman method development to monitor and guide the expedited biologics process development.

Two Multidimensional Chromatography/High-Resolution Mass Spectrometry Approaches Enabling the In-Depth Metabolite Characterization Simultaneously from Three Glycyrrhiza Species: Method Development, Comparison, and Integration.

Two offline multidimensional chromatography/high-resolution mass spectrometry systems (method 1: fractionation and online two-dimensional liquid chromatography, 2D-LC; method 2: fractionation and offline 2D-LC) were established to characterize the metabolites simultaneously from three Glycyrrhiza species. Ion exchange chromatography in the first-dimensional (1D) separation was well fractionated between the acidic (mainly triterpenoids) and weakly acidic components (flavonoids). These obtained subsamples got sophisticated separation by the second (2D) and third dimension (3D) of chromatography either by online reversed-phase chromatography × reversed-phase chromatography (RPC × RPC) or offline hydrophilic interaction chromatography × RPC (HILIC × RPC). Orthogonality for the 2D/3D separations reached 0.73 for method 1 and 0.81 for method 2, respectively. We could characterize 1097 compounds from three Glycyrrhiza species based on an in-house library and 33 reference standards, involving 618 by method 1 and 668 by method 2, respectively. They exhibited a differentiated performance and complementarity in identifying the multiple subclasses of Glycyrrhiza components.

Two-dimensional high performance liquid chromatography purification of underivatized urinary prednisone and prednisolone for compound-specific stable carbon isotope analysis.

The gas chromatography-combustion isotope ratio mass spectrometry (GC/C/IRMS) confirmation procedure for prednisone (PS) and prednisolone (PSL) is still a great challenge for the doping control laboratory due to the many structurally similar steroids present in urinary matrices. This study aims to establish an innovative online two-dimensional high performance liquid chromatography (2D-HPLC) purification method for measuring the carbon isotope ratios (CIRs) and achieving the identification of the synthetic forms of these two endogenous anabolic androgenic steroids (EAASs). Initially, the one-dimensional chromatographic column was used to separate and purify endogenous reference compounds (ERCs), and the co-elution fluids containing PS and PSL were switched to a two-dimensional chromatographic column for further purification through an online transfer system. Then the purified compounds were analyzed using GC/C/IRMS after sample pretreatments. The results showed that the minimum detection concentration of PS and PSL reached 30 ng mL-1, and no isotope fractionation occurred during the entire collection and preparation process. This method has been validated with the WADA technical document and showed good sensitivity and selectivity, demonstrating its practical applicability for urine samples in doping control laboratories.

Characterization of Dye-Loaded Poly(lactic-co-glycolic acid) Nanoparticles by Comprehensive Two-Dimensional Liquid Chromatography Combining Hydrodynamic and Reversed-Phase Liquid Chromatography.

Analytical methods for the assessment of drug-delivery systems (DDSs) are commonly suitable for characterizing individual DDS properties, but do not allow determination of several properties simultaneously. A comprehensive online two-dimensional liquid chromatography (LC × LC) system was developed that is aimed to be capable of characterizing both nanoparticle size and encapsulated cargo over the particle size distribution of a DDS by using one integrated method. Polymeric nanoparticles (NPs) with encapsulated hydrophobic dyes were used as model DDSs. Hydrodynamic chromatography (HDC) was used in the first dimension to separate the intact NPs and to determine the particle size distribution. Fractions from the first dimension were taken comprehensively and disassembled online by the addition of an organic solvent, thereby releasing the encapsulated cargo. Reversed-phase liquid chromatography (RPLC) was used as a second dimension to separate the released dyes. Conditions were optimized to ensure the complete disassembly of the NPs and the dissolution of the dyes during the solvent modulation step. Subsequently, stationary-phase-assisted modulation (SPAM) was applied for trapping and preconcentration of the analytes, thereby minimizing the risk of analyte precipitation or breakthrough. The developed HDC × RPLC method allows for the characterization of encapsulated cargo as a function of intact nanoparticle size and shows potential for the analysis of API stability.

Comprehensive online two-dimensional liquid chromatography coupled to hybrid linear ion trap Orbitrap mass spectrometry with optimized dilution and modulation for analysis of Huangqi Guizhi Wuwu decoction.

Huangqi Guizhi Wuwu decoction (HGWD), as a classical formula, has been used for thousands of years in China. In this work, a comprehensive strategy was proposed for characterizing the chemical profile of HGWD based on online two-dimensional hydrophilic interaction and reversed-phase liquid chromatography coupled with hybrid linear ion trap-Orbitrap mass spectrometry (online HILIC × RP-ESI/HRMS/MSn). The compounds in HGWD were first separated by the combined use of an XBridge amide column (150 × 4.6mm, 3.5μm) and Accucore C18 column (50mm × 4.6mm, 2.6μm). Modulation with assistant technology, including trap columns and online dilution, was optimized and developed to decrease potential analyte loss and improve the resolution of the system. Subsequently, the accurate mass was determined by high-resolution Orbitrap and MSn fragment data by a hybrid linear ion trap (LTQ). In total, 170 chemical constituents were unambiguously identified or tentatively characterized in both positive and negative ion modes. Our study demonstrated that the proposed online HILIC × RP system coupled to the LTQ-Orbitrap MS platform is an efficient analytical technique for characterizing the chemical profile of multicomponent systems.

Screening strategy for 1210 exogenous chemicals in serum by two-dimensional liquid chromatography-mass spectrometry

Humans are at risk of exogenous exposure to exogenous chemicals. Challenges exist for the comprehensive monitoring of residues with different physical and chemical properties in serum. Here, an on-line two-dimensional liquid chromatography (2D-LC) - high resolution mass spectrometry system (HRMS) was developed, expanding the range of the partition coefficient in octanol/water of the residue analysis from −8 to 12. A high-coverage serum residue screening strategy was further designed by integrating 2D-LC system with HRMS full MS/data independent acquisition and automatic spectral library searching. This strategy enables to simultaneously screen 1210 pesticides, veterinary/human drugs, other chemical pollutants and their metabolites in serum with a single analysis. Method validation showed 92% and 81% of 1022 residues spiked in serum could be detected at 50 ng/mL and 5 ng/mL, respectively. The developed method was applied to the analysis of 24 separately pooled serum samples, 58 suspect residues were found, some of them were detected at high frequencies over than 50%. Among them, 4,6-Dinitro-O-cresol and probable carcinogenic folpet are highly toxic, and cimaterol is banned in China. Collectively, this study developed a 2D-LC-HRMS -based screening strategy for screening pesticides, veterinary/human drugs, and other chemical pollutants in serum, it is helpful for studying the effect of exogenous exposures on human health.

Development of an On-Line Two-dimensional Normal Phase Liquid Chromatography System for Analysis of Weakly Polar Samples

In this study, a novel on-line two-dimensional (2D) normal phase × normal phase liquid chromatography (NPLC × NPLC) was developed for the separation of weakly polar samples. The 2D NPLC was integrated by a unique designed solvent evaporation (SE) interface, including a 6-port 2-position valve and a 10-port 2-position valve in conjunction with two silica gel-packed enrichment columns. The enrichment columns played a pivotal role in evaporating normal phase (NP) solvent from the first dimensional NPLC under vacuum and elevated temperature condition. The working parameters of the interface were evaluated comprehensively. To demonstrate the resolving powerful 2D system, we analyzed three natural-sourced weakly polar compounds from the extracts of toad venom, dammar resin, and propolis. To this end, we studied the effects of mobile phase combinations, the second dimensional column length for separation of the extracts from toad venom, and propolis, respectively. In general, we have found that excellent separation can be obtained with diversity of NP solvent combinations and longer NPLC column (150 mm long). Moreover, compared with the generally used 2D reversed-phase liquid chromatography (RPLC × RPLC), the NPLC × NPLC method exhibited better separation orthogonality. In conclusion, the new NPLC × NPLC separation method provides potential advantages for analysis of weakly polar samples.

Optimizing transfer and dilution processes when using active solvent modulation in on-line two-dimensional liquid chromatography

Two-dimensional liquid chromatography (2D-LC) is becoming increasingly popular for the analysis of complex samples, which is partly due to the recent introduction of commercial 2D-LC systems. To deal with the mobile phase incompatibility between highly orthogonal retention mechanisms, such as hydrophilic interaction liquid chromatography (HILIC) and reversed-phase LC (RPLC), several strategies have been introduced over the years. One of these strategies is active solvent modulation (ASM), a valve-based approach allowing the on-line dilution of the effluent eluting from the first dimension before transfer to the second dimension. This strategy has gained a lot of attention and holds great potential, however, no clear guidelines are currently in place for its use. Therefore, this study aims to investigate how the ASM process can be optimized when using highly incompatible LC combinations, such as HILIC and RPLC, in a simplified selective comprehensive 2D-LC set-up (sHILIC x RPLC) to suggest guidelines for future users. Using a representative sample, the dilution factor (DF), the duration of the ASM phase, the filling percentage of the sample loops, and their unloading configuration are investigated and optimized. It is observed that a DF of 10 with an optimal ASM phase duration, a sample loop filling of maximum 25%, and an unloading configuration in backflush mode, result in the best peak shapes, intensities, and recoveries for early eluting compounds, while keeping the total analysis time minimal. Based on these results, some general recommendations are made that could also be applied in other 2D-LC modes, such as comprehensive 2D-LC (LC x LC), heart-cutting 2D-LC (LC–LC), and other chromatographic combinations with mobile phase incompatibility issues.

Urinary Measurement of Epigenetic DNA Modifications and 8-oxodG as Possible Noninvasive Markers of Colon Cancer Evolution.

The active DNA demethylation mechanism involves 5-methylcytosine (5-mCyt) enzymatic oxidation with the subsequent formation of 5-hydroxymethylcytosine (5-hmCyt), which can be further oxidized to 5-formylcytosine (5-fCyt) and 5-carboxylcytosine (5-caCyt). The products of active DNA demethylation are released into the bloodstream and eventually also appear in urine. We used online two-dimensional ultraperformance liquid chromatography with tandem mass spectrometry (2D-UPLC-MS/MS) to compare DNA methylation marks and 8-oxo-2'-deoxyguanosine (8-oxodG) in colorectal cancer and pre-cancerous condition in urine. The study included four groups of subjects: healthy controls, patients with inflammatory bowel disease (IBD), persons with adenomatous polyps (AD), and individuals with colorectal cancer (CRC). We have found that the level of 5-fCyt in urine was significantly lower for CRC and polyp groups than in the control group. The level of 5-hmCyt was significantly higher only in the CRC group compared to the control (2.3 vs. 2.1 nmol/mmol creatinine). Interestingly, we have found highly statistically significant correlation of 5-hydroxymethyluracil with 5-hydroxymethylcytosine, 5-(hydroxymethyl)-2'-deoxycytidine, 5-(hydroxymethyl)-2'-deoxyuridine, and 5-methyl-2'-deoxycytidine in the CRC patients' group.

Novel Online Three-Dimensional Separation Expands the Detectable Functional Landscape of Cellular Phosphoproteome.

Protein phosphorylation is a prevalent post-translational modification that regulates essentially every aspect of cellular processes. Currently, liquid chromatography-tandem mass spectrometry (LC-MS/MS) with an extensive offline sample fractionation and a phosphopeptide enrichment method is a best practice for deep phosphoproteome profiling, but balancing throughput and profiling depth remains a practical challenge. We present an online three-dimensional separation method for ultradeep phosphoproteome profiling that combines an online two-dimensional liquid chromatography separation and an additional gas-phase separation. This method identified over 100,000 phosphopeptides (>60,000 phosphosites) in HeLa cells during 1.5 days of data acquisition, and the largest HeLa cell phosphoproteome significantly expanded the detectable functional landscape of cellular phosphoproteome.

Characterization and comparison of smokeless powders by on-line two-dimensional liquid chromatography

Smokeless powders (SPs) are one of the most commonly used propellants for ammunition but can also be abused as energetic material in improvised explosive devices (IEDs) such as pipe bombs. After a shooting or explosion, unburnt or partially burnt particulates may be observed which can be used for forensic investigation. SPs comprise mainly nitrocellulose (NC) and additives. Therefore, the characterization of both NC and the additives is of significant forensic importance. Typically, the identification, classification, and chemical profiling of smokeless powders are based exclusively on the analysis of the additives. In this study, information regarding the NC base component was combined with the chemical analysis of the additives using two-dimensional liquid chromatography (2D-LC). The system combines size-exclusion chromatography (SEC) and reversed-phase liquid chromatography (RPLC) in an on-line heart-cut 2D-LC configuration. In the first dimension, the NC is characterized by its molecular-weight distribution (MWD) while being separated from the additives. The additives are then transferred to the second-dimension separation using a novel analyte-transfer system. In the second dimension, the additives are separated to obtain a detailed profile of the low-molecular-mass compounds in the SP. With this approach, the MWD of the NC and the composition of the additives in SP have been obtained within an hour. A discrimination power of 90.53% was obtained when studying exclusively the NC MWD, and 99.47% for the additive profile. This novel combination enables detailed forensic comparison of intact SPs. Additionally, no extensive sample preparation is required, making the developed method less labor intensive.

Development of a sub-hour on-line comprehensive cation exchange chromatography x RPLC method hyphenated to HRMS for the characterization of lysine-linked antibody-drug conjugates.

This study details the development of on-line two-dimensional liquid chromatography (2D-LC) methods combining cation-exchange chromatography (CEX) and reversed-phase liquid chromatography (RPLC) for the separation of the charge variants of a lysine-linked antibody-drug conjugate (ADC). This combination gives an excellent example of the potential benefits of 2D-LC approaches for the analysis of such complex protein formats. CEX is considered the reference technique for the separation of protein charge variants but its retention mechanism usually requires the use of a high concentration of non-volatile salts, which impedes its compatibility with MS detection. In this context, the use of an on-line 2D-LC-MS approach not only allows on-line desalting and indirect coupling of CEX with mass spectrometry (MS) detection but it also provides increased and complementary information within a single analysis. The first part of this study was devoted to the choice of stationary phases and the optimization of chromatographic conditions in both dimensions. Based on the results obtained in 1D-CEX with ultraviolet detection (UV) and 1D-RPLC with UV and high-resolution mass spectrometry (HRMS) detections, an on-line comprehensive two-dimensional liquid chromatography method combining CEX and RPLC was developed. The last part of this study was devoted to the identification of the separated species using HRMS detection and in the comparison of three ADC samples exposed to different durations of thermal stress.

Proteomic signatures for perioperative oxygen delivery in skin after major elective surgery: mechanistic sub-study of a randomised controlled trial

BackgroundMaintaining adequate oxygen delivery (DO2) after major surgery is associated with minimising organ dysfunction. Skin is particularly vulnerable to reduced DO2. We tested the hypothesis that reduced perioperative DO2 fuels inflammation in metabolically compromised skin after major surgery. MethodsParticipants undergoing elective oesophagectomy were randomised immediately after surgery to standard of care or haemodynamic therapy to achieve their individualised preoperative DO2. Abdominal punch skin biopsies were snap-frozen before and 48 h after surgery. On-line two-dimensional liquid chromatography and ultra-high-definition label-free mass spectrometry was used to characterise the skin proteome. The primary outcome was proteomic changes compared between normal (≥preoperative value before induction of anaesthesia) and low DO2 (<preoperative value before induction of anaesthesia) after surgery. Secondary outcomes were functional enrichment analysis of up/down-regulated proteins (Ingenuity pathway analysis; STRING Protein-Protein Interaction Networks). Immunohistochemistry and immunoblotting confirmed selected proteomic findings in skin biopsies obtained from patients after hepatic resection. ResultsPaired punch skin biopsies were obtained from 35 participants (mean age: 68 yr; 31% female), of whom 17 underwent oesophagectomy. There were 14/2096 proteins associated with normal (n=10) vs low (n=7) DO2 after oesophagectomy. Failure to maintain preoperative DO2 was associated with upregulation of proteins counteracting oxidative stress. Normal DO2 after surgery was associated with pathways involving leucocyte recruitment and upregulation of an antimicrobial peptidoglycan recognition protein. Immunohistochemistry (n=6 patients) and immunoblots after liver resection (n=12 patients) supported the proteomic findings. ConclusionsProteomic profiles in serial skin biopsies identified organ-protective mechanisms associated with normal DO2 after major surgery. Clinical trial registrationISRCTN76894700.

Strategy for high-throughput identification of protein complexes by array-based multi-dimensional liquid chromatography-mass spectrometry

Comprehensive elucidation of the composition of multiprotein complexes in model organisms is essential to understand conserved biological systems, but large-scale mapping physical association networks is still challenging due to limited throughput of present methods. In this work, a strategy coupling array-based online two-dimensional liquid chromatography (array-based 2D-LC) and liquid chromatography-tandem mass spectrometry (LC-MS/MS) was demonstrated for high throughput and in-depth identification of protein complexes from cultured human HeLa cell extracts. Mixed-bed ion-exchange column was employed as the first dimensional (1stD) separating mode and an array consisting of eight reversed phase columns was developed as the second dimensional (2ndD) mode. Taking advantage of array parallel strategy, this online system showed an 8-fold increase in throughput. After array-based online 2D-LC separation, altogether 256 × 2ndD fractions were collected for further LC-MS/MS analysis. Public databases of protein-protein interaction (PPI) and co-elution curves identified by LC-MS were applied to reconstruct the protein complexes. A rigorous inspection was operated by cataloging the protein complexes into chromatographic fractions to minimize the number of false positives. As result, a total number of 4,436 proteins were identified and 26,092 elution curves were graphed. A network consisting of 47,745 PPIs was established among 2,201 proteins and presented 1,530 putative protein complexes with high confidence. Most of the identified PPIs were linked to diverse biological processes and may reveal further disease mechanism and therapeutic strategy.

On-line two-dimensional liquid chromatography hyphenated to mass spectrometry and ion mobility-mass spectrometry for the separation of carbohydrates from lignocellulosic biomass

Lignocellulosic biomass is a promising resource of renewable energy. Its transformation to ethanol requires efficient pretreatment leading to complex liquid mixtures made of hundreds of oxygenated analytes. A large part of the released compounds belong to the carbohydrates family. To overcome the complexity of such samples, a comprehensive on-line two-dimensional reversed-phase liquid chromatography hyphenated to high-resolution mass spectrometry (RPLC × RPLC-HRMS) was dedicated to the separation of carbohydrates and more specifically oligomers coming from pretreated lignocellulosic biomass. The first part of this study consisted in the optimization of such hyphenation (i.e. selection of stationary phases, mobile phases, sampling time, etc.). Then, the analytical method was applied to an industrial aqueous biomass product coming from the sulfuric acid-based pretreatment of a wheat straw. Around 70 well-resolved chromatographic peaks corresponding to oligomers were obtained. Occupation of the separation space between each chromatographic dimension was estimated to 75%. In the last part of this study, the interest of ion mobility-mass spectrometry in addition to RPLC × RPLC was discussed. Some examples highlighted the additional separation that can bring ion mobility to RPLC × RPLC-IMS-HRMS method. Using this four-dimensional hyphenation method, each analyte was described by two retention times, the collisional cross section and the molecular formula allowing to reach a level of detail never seen for biomass sample compositions.

Investigation of cardiolipin oxidation products as a new endpoint for oxidative stress in C. elegans by means of online two-dimensional liquid chromatography and high-resolution mass spectrometry

The investigation of neurodegenerative and age-related diseases is a highly relevant topic in current research. Especially oxidative stress is thought to be the common underlying mechanism in diseases such as Parkinson’s or Alzheimer’s disease. The nematode Caenorhabditis elegans (C. elegans) is a prominent model organism, which is often used for such investigations and has gained extensive recognition in research regarding the linkage of reactive oxygen species (ROS) and neurodegeneration. Not only studies regarding genomics and proteomics have been increasingly conducted, also the number of studies based on the lipidome is rising. The phospholipid class of cardiolipin (CL) is a unique lipid class, which is exclusively located in mitochondria and is therefore of great relevance regarding oxidative stress and associated diseases. CL oxidation products have become a prominent marker for oxidative stress in various organisms. However, the CL distribution in the nematode C. elegans is still scarcely known on the molecular level and oxidation products have not yet been identified. In this work, we demonstrate the importance of CL distribution and the applicability of CL oxidation products as a sensitive marker for oxidative stress in C. elegans. For this reason, the CL distribution was determined by means of online two-dimensional liquid chromatography hyphenated with high-resolution mass spectrometry (2D-LC/HRMS). Subsequently, worms were treated with tert-butyl hydroperoxide (tBOOH) in order to provoke oxidative stress and induce the artificial formation of oxidized CL. We were able to detect increasing amounts of CL oxidation products of highly unsaturated CL species in a concentration-dependent manner. This finding emphasizes the great potential of CL oxidation products as a sensitive marker substance of oxidative stress in C. elegans, which is not only directly linked to mitochondria function but also favourable to other oxidative stress markers in terms of the needed sample material, relative substance stability and specificity of the oxidation site.

Quantification of DNA Modifications Using Two-Dimensional Ultraperformance Liquid Chromatography Tandem Mass Spectrometry (2D-UPLC-MS/MS).

Our hereby presented methodology is suitable for reliable assessment of the most common DNA modifications which arise as a product of fundamental metabolic processes. 8-oxoguanine, one of the oxidatively modified DNA bases is a typical biomarker of oxidative stress. A noncanonical base, uracil, may also be present in small quantities in DNA. Ten-eleven translocation (TET) proteins are involved in oxidation of 5-methylcytosine to 5-hydroxymethylcytosine which can be further oxidized to 5-formylcytosine and 5-carboxycytosine. 5-hydroxymethyluracil may be formed in deamination reaction of 5-hydroxymethylcytosine or can also be generated by TET enzymes. All the above mentioned modifications seem to play some regulatory roles. Here, we provide a protocol for isotope-dilution automated online two-dimensional ultraperformance liquid chromatography with tandem mass spectrometry (2D-UPLC-MS/MS) for direct measurement of 5-methyl-2'-deoxycytidine, 5-(hydroxymethyl)-2'-deoxycytidine, 5-formyl-2'-deoxycytidine, 5-carboxy-2'-deoxycytidine, 5-(hydroxymethyl)-2'-deoxyuridine, 2'-deoxyuridine, and 8-oxo-2'-deoxyguanosine. We also provide optimized protocols for extraction of DNA, fully compatible with the downstream MS/MS analysis.