Capillary Chromatography Research Articles

L-Cysteine-Bonded Polymeric Monolithic Stationary Phase for Enantioseparation of Dansyl Amino Acids in Capillary Liquid Chromatography.

A chiral monolith stationary phase was fabricated by modifying the monolith surface using L-cysteine through a thiol-epoxy click reaction. L-cysteine-bonded polymer monolith was characterized by scanning electron microscopy/energy-dispersive X-ray and attenuated total reflectance Fourier-transformed infrared. The monomer content and modification temperature were carefully optimized to create a polymer monolith with excellent mechanical stability and permeability. Our findings revealed that the column morphology depended significantly on the porogen concentration and modification temperature for its morphology and efficiency. Adequate pores and binding sites were formed with the optimal porogen content, while a higher modification temperature improved the modification yield, enhancing peak shapes and increasing separation efficiency. The column demonstrated its capability for enantioseparation of dansyl glutamic acid, dansyl aspartic acid, dansyl methionine, and dansyl phenylalanine using a 60mM ammonium acetate buffer solution and acetonitrile in a 20:80 v/v ratio. It maintained good mechanical stability and repeatability with no relative standard deviation exceeding 7%. These results indicated that the L-cysteine-bonded polymer monolith has excellent potential as a chiral stationary phase.

Considerations for method development and method translation in capillary liquid chromatography: A tutorial

HPLC remains one of the most widely used measurement techniques for chemical analysis. Capillary LC, which utilizes narrow diameter columns operated at lower flow rates than analytical-scale LC, continues to gain adoption based on its reduced mobile phase consumption and increased sensitivity when coupled to MS detection. This tutorial offers practical insights into the most critical aspects of translating analytical-scale separations to the capillary scale. The selection of pumping systems, detectors, and the potential for performance loss due to extra-column effects are examined within the context of separations using columns with inner diameters ≤ 0.3 mm. Column choices within this diameter range are also detailed, both in terms of stationary phase support options and general commercial availability. The impact of these various factors on the effective development/translation of LC methods down to flow rates under 10 µL/min is described to provide readers with a basis for implementing these strategies within their own analytical workflows.

Development of microflow ultra high performance liquid chromatography-mass spectrometry metabolomic assays for analysis of mammalian biofluids

Introduction and objectivesThe application of untargeted metabolomics assays using ultra high performance liquid chromatography-mass spectrometry (UHPLC-MS) to study metabolism in biological systems including humans is rapidly increasing. In some of these studies there is a requirement to collect and analyse low sample volumes of biofluids (e.g. tear fluid) or low cell and tissue mass samples (e.g. tissue needle biopsies). The application of microflow, capillary or nano liquid chromatography (≤ 1.0 mm column internal diameter (i.d.)) theoretically should accomplish a higher assay sensitivity compared to analytical liquid chromatography (2.1–5.0 mm column internal diameter). To date, there has been limited research into microflow UHPLC-MS assays that can be applied to study samples of low volume or mass.MethodsThis paper presents three complementary UHPLC-MS assays (aqueous C18 reversed-phase, lipidomics C18 reversed-phase and Hydrophilic Interaction Liquid Chromatography (HILIC)) applying 1.0 mm internal diameter columns for untargeted metabolomics. Human plasma and urine samples were applied for the method development, with porcine plasma, urine and tear fluid used for method assessment. Data were collected and compared for columns of the same length, stationary phase and stationary phase particle size but with two different column internal diameters (2.1 mm and 1.0 mm).Results and conclusionsAll three assays showed an increase in peak areas and peak widths when applying the 1.0 mm i.d. assays. HILIC assays provide an advantage at lower sample dilutions whereas for reversed phase (RP) assays there was no benefit added. This can be seen in the validation study where a much higher number of compounds were detected in the HILIC assay. RP assays were still appropriate for small volume samples with hundreds of compounds being detected. In summary, the 1.0 mm i.d. column assays are applicable for small volume samples where dilution is required during sample preparation.

Effects of Zinc Phthalocyanine Photodynamic Therapy on Vital Structures and Processes in Hela Cells.

This work presents results on the efficiency of newly designed zinc phthalocyanine-mediated photodynamic therapy of both tumoral and nontumoral cell models using the MTT assay. Further detailed examinations of mechanistic and cell biological effects were focused on the HELA cervical cancer cell model. Here, ROS production, changes in the mitochondrial membrane potential, the determination of genotoxicity, and protein changes determined by capillary chromatography and tandem mass spectrometry with ESI were analyzed. The results showed that, in vitro, 5 Jcm-2 ZnPc PDT caused a significant increase in reactive oxygen species. Still, except for superoxide dismutase, the levels of proteins involved in cell response to oxidative stress did not increase significantly. Furthermore, this therapy damaged mitochondrial membranes, which was proven by a more than 70% voltage-dependent channel protein 1 level decrease and by a 65% mitochondrial membrane potential change 24 h post-therapy. DNA impairment was assessed by an increased level of DNA fragmentation, which might be related to the decreased level of DDB1 (decrease in levels of more than 20% 24 h post-therapy), a protein responsible for maintaining genomic integrity and triggering the DNA repair pathways. Considering these results and the low effective concentration (LC50 = 30 nM), the therapy used is a potentially very promising antitumoral treatment.

B-169 Combining the EXENT System and LC-MS for the Detection of Monoclonal Immunoglobulins at Low Levels

Abstract Background The EXENT® system is an automated platform designed to quantify monoclonal immunoglobulins in serum. EXENT® combines immunoprecipitation and MALDI-TOF mass spectrometry for the detection of monoclonal immunoglobulins at levels lower than traditional gel based methods. This study was designed to demonstrate if EXENT® prepared samples that were negative could be reflexed to a more sensitive method based on capillary flow Liquid Chromatography- Mass Spectrometry (LC-MS) without any modifications to the EXENT® prepared sample. Methods Patient samples containing a monoclonal immunoglobulin (ranging from 20 to 40 g/L) were diluted into pooled polyclonal serum and the samples were prepared and analyzed by EXENT®. Samples that were negative by EXENT® were reflexed by directly loading EXENT® eluates onto the LC-MS instrument. The abundance of the monoclonal immunoglobulin was defined by the signal-to-noise (S/N) of the light chain peak observed after deconvolution. Results Baseline samples were diluted down to two levels, 0.100 g/L and 0.001 g/L. LC-MS detected the same light chain from the monoclonal immunoglobulin as the EXENT® system in all reflexed samples that were negative by EXENT®. Conclusions LC-MS can detect monoclonal immunoglobulins that can no longer be detected using the EXENT® system by tracking the unique molecular mass of the monoclonal light chain. Reflexing samples to LC-MS did not require additional sample handling resulting in a faster time-to-result than current NGS, NGF, and clonotypic peptide approaches. Furthermore, monitoring the intact monoclonal light chain circumvents enzymatic cleavage to create a unique clonotypic peptide, alleviating a situation where no unique peptide can be generated as has been shown previously by Bergen et al. As a consequence, this reflex-methodology results in a consistent way of measuring the presence of malignant B-cells with high sensitivity.

The rapid detection of pesticide residues in fruits and vegetables using electrochemical methods

To meet global demand, pesticides play a very important role in the control of pests that damage agricultural products. However, overuse of pesticides threatens ecosystems and human health. In recent years, gas chromatography, liquid chromatography, gas chromatography-mass spectrometry, liquid chromatography-mass spectrometry, capillary chromatography and capillary electrophoresis have been used in the determination of pesticides. Although these methods are highly specific, highly selective and sensitive, the high value of sophisticated laboratory equipment and tools is a complex and time-consuming process. Because of this, electrochemical sensors and biosensor platforms have become good analytical methods for detecting pesticides due to a number of advantages, including ease of detection of system manufacturing processes, high sensitivity and selectivity. This article describes the methods of manufacturing portable electrochemical sensors for detecting pesticide residues in fruits and vegetables on site, that is, the achievements in recent years in the field of cyclic voltammetry, square wave voltammetry, differential pulse voltammetry and electrochemical impedance spectroscopy. In addition, detailed information is provided on the basic mechanism and electrochemical sensitivity of these methods. The development of electrochemical methods and real-time monitoring of smartphone technologies, as well as their integration with detection platforms, are discussed. For readers with a scientific and technological focus, this article will provide additional valuable information for understanding the creation of portable electrochemical devices, rapid detection of pesticides, the role of electrochemical sensitive methods and contributing to their further development.

Microdialysis-integrated HPLC system with dual-electrode detection using track-etched membrane electrodes for in vivo monitoring of dopamine dynamics

A capillary high-performance liquid chromatography (HPLC) system equipped with a dual-electrode detector utilizing track-etched membrane electrodes (TEMEs) was combined with a microdialysis sampling setup. The electrochemical detector benefits from the high electrolysis efficiency of TEMEs, allowing for calibration-free coulometric detection and simplifying data analysis to determine the dopamine recovery through a dialysis probe. Additionally, this system was used for in vivo monitoring of dopamine in the right striatum of a mouse brain. Temporal changes in dopamine levels, including an exponential decay immediately after the dialysis probe insertion and an excess release of dopamine induced by a high concentration of potassium ions, confirmed the system’s proper operation. Furthermore, subsequent measurements following the intraperitoneal injection of mirtazapine showed no increase in dopamine levels in the right dorsal striatum. The dual-electrode system displayed characteristic dopamine detection behavior, with anodic and cathodic peak pairs indicative of reversible electrochemical reactions. This capability facilitated the identification of the dopamine peak within the complex chromatogram of the mouse brain dialysate. The consistency between dopamine collection efficiency from standard solutions and dialysate indicated the absence of interfering electroactive substances overlapping with the dopamine peak in the chromatogram. This integrated analysis system successfully tracked temporal fluctuations in dopamine concentration within the mouse brain.

A stationary phase of tetraethylene glycol-modified silica for separation of phenolic acids

AbstractSilica, as a stationary phase, has low separation efficiency accompanied by overlapping, broadened, and tailed peaks, so it needs to be modified to improve its efficiency. This study aims to develop a silica-based stationary phase modified by tetraethylene glycol (TEG) to separate phenolic compounds. Silica was modified by a chemical bond between silanol groups on the silica surface and TEG through a 3-glycidyloxypropylmethoxysilane reaction. The modified silica was packed into a capillary column and used to separate simple phenolic compounds consisting of phenol, pyrocatechol, and pyrogallol. A sample of 0.2 µL was injected into the capillary liquid chromatography and the mobile phase employed was acetonitrile 98% with a flow rate of 3 μL min−1. Elution was also done isocratically in this process and detection was carried out at a wavelength of 254 nm. The mixture of simple phenolic compounds was successfully separated in less than 7 min. The asymmetry factor and resolution were 1.43–2.12 and 1.72–5.43 respectively. The number of the theoretical plates ranged from 213 to 7,857. Columns containing Si-TEG stationary phase also separate phenolic compounds, which consist of gallic acid, syringic acid, ferulic acid, and caffeic acid. A sample of 0.2 µL was injected into the capillary liquid chromatography and successfully separated the mixture in less than 12 min. The samples were eluted isocratically using a mixture of methanol and 50 mM phosphate buffer pH 2.5 (8:92) with a flow rate of 3 μL min−1. The phenolic acids compounds were detected at a wavelength of 280 nm. The chromatogram showed four separate peaks. The asymmetry factor and resolution were 1.53–1.63 and 1.14–1.74, respectively, but the number of the theoretical plates was low, ranging from 190 to 796.

Analysis of doxorubicin and fullerenol in rat serum by micellar electrokinetic capillary chromatography.

A new micellar electrokinetic capillary chromatographic (MEKC) method has been developed and optimized for simultaneous quantitation of doxorubicin (Dox) and fullerenol (Frl) in rat serum. The separation was carried out in a capillary (48.5-40 cm to the detector - 50 µm id fused-silica capillary with bubble cell, 150 µm) at an applied voltage of 25 kV and temperature of 25 °C. For the background electrolyte 10 mmol L- 1 borate buffer pH 9.3 plus 15 mmol L-1 phosphate buffer pH 7.0 (with the final pH of the mixture adjusted to 7.0 with HCl), with added 10 % (V/V) methanol, and 15 mmol L-1 sodium dodecyl sulfate as a surfactant, were used. The hydrodynamic injection was carried out at 5.0 kPa during the period of 100 s. Linear calibration curves were established over the concentration range 0.5-500.0 mg L- 1 for Dox and 10.0-500.0 mg L- 1 for Frl (at 234 nm). The proposed MEKC procedure was fully validated and applied for the deter mination of Dox and Frl in Wistar rats after intra pe ritoneal administration of both molecules.

Immobilised artificial membrane liquid chromatography vs liposome electrokinetic capillary chromatography: Suitability in drug/bio membrane partitioning studies and effectiveness in the assessment of the passage of drugs through the respiratory mucosa

This study pioneers a comparison of the application of biomimetic techniques, immobilised artificial membrane liquid chromatography (IAM LC) and liposome electrokinetic capillary chromatography (LEKC), for the prediction of pulmonary drug permeability. The pulmonary absorption profiles of 26 structurally unrelated drug-like molecules were evaluated using their IAM hydrophobicity index (CHI IAM) measured in IAM LC, and the logarithm of distribution constants (log KLEKC) derived from the LEKC experiments. Lipophilicity (phospholipids) parameters obtained from IAM LC and most LEKC analyses were linearly related to the n-octanol/water partitioning coefficients of the neutral forms (i.e., log Po/w values) to a moderate extent. However, the relationship with distribution coefficients at the experimental pH (7.4) (i.e., log D7.4) were weaker overall for IAM LC data and sigmoidal for some liposome compositions (phosphatidyl choline (PC): phosphatidyl inositol (PI) 85:15 mol% and 90:10 mol%) and concentrations (4 mM) in LEKC. This suggests that phospholipid partitioning supports both hydrophobic and electrostatic interactions occurring between ionised drugs and charged phospholipid moieties. The latter interactions are original when compared to those taking place in the more established n-octanol/water partitioning systems.A stronger correlation (R2 > 0.65) was identified between the LEKC retention parameters, and the experimental apparent lung permeability (i.e., log Papp values) as opposed to the values obtained by IAM LC. Therefore, LEKC offers unprecedented advantages over IAM LC in simulating cell membrane partitioning processes in the pulmonary delivery of drugs. Although LEKC has the advantage of more effectively simulating the electrostatic and hydrophobic forces in drug/pulmonary membrane interactions in vitro, the technique is unsuitable for analysing highly hydrophilic neutral or anionic compounds at the experimental pH. Conversely, IAM LC is useful for analysing compounds spanning a wider range of lipophilicity. Its simpler and more robust implementation, and propensity for high-throughput automation make it a favourable choice for researchers in drug development and pharmacological studies.

Hybrid organic monolithic column containing MIL-68(Al) for the separation of small molecules by capillary HPLC

A hybrid organic monolithic column made of poly(lauryl methacrylate-co-1,6-hexanediol dimethacrylate) and the metal-organic framework MIL-68(Al) was prepared for the first time. The column was used in capillary liquid chromatography, both in isocratic and gradient elution modes. Separation performance towards small molecules of different chemical nature (polycyclic aromatic hydrocarbons, alkylbenzenes, phenols, etc.) was studied. Monte Carlo simulations were made to both select the proper precursors to obtain empty metal-organic framework micropores in the monolithic polymer and also, to analyze the potential free access of the studied analytes into the micropores (necessary to improve mass transfer and column efficiency). The hereby synthesized metal-organic framework microcrystals allowed obtaining homogeneous hybrid monolithic columns. Adding of MIL-68(Al) (1030 m2 g-1 BET specific surface area) increased the surface area from 3.9 m2 g-1 for the parent monolith to 18.2 m2 g-1 for the hybrid column containing 8 mg mL-1 of the microcrystals. Chromatographic performance of this new column was evaluated by studying retention factors, resolution, and plate counts at room temperature. Different compounds, not completely resolved in the parent monolith, were partially or completely separated after metal-organic framework addition. Using the monolithic column with only 2 mg mL-1 of MIL-68(Al), five alkylbenzenes were completely separated with very symmetrical peak shapes, resolution factors up to 3.60 and plate counts of 4300 plates m-1 for n-hexylbenzene. This value is higher than those obtained by other authors who used organic monolithic columns with embedded metal-organic frameworks to perform separations at room temperature. Additionally, nine polycyclic aromatic hydrocarbons were partially or completely resolved in gradient elution mode. The hybrid monolithic columns exhibited very good intra-day (%RSD=1.9), inter-day (%RSD=2.6), and column-to-column (%RSD=4.3) reproducibility values. Easy and fast column preparation, and versatility to efficiently separate several compounds of different chemical nature in isocratic and gradient mode, makes this new hybrid column a very good option for the analysis of small molecules in capillary (or nano) HPLC.

Development of a capillary electrophoresis method with laser-induced fluorescence detection for the characterization of oligosaccharides in human milk

Breast milk is the most appropriate food for an infant in the first months of life - it provides the essential nutrients required for proper growth, with breast milk oligosaccharides (HMOs) being the third most abundant component. HMO concentrations vary based on factors like lactation stage and maternal health. They play crucial roles in infant health, acting as prebiotics, antimicrobials, and immune modulators. Analytical methods like capillary electrophoresis and liquid chromatography are the most often used in HMOs qualitative and quantitative analysis. Continuing research aims to enhance analytical techniques for comprehensive HMOs analysis, this work presents the development of a capillary electrophoresis method with laser-induced fluorescence detection along with derivatization and solid-phase extraction steps for the characterisation of major oligosaccharides in colostrum samples. Validation parameters, such as linearity (0.9949–0.9989), limits of detection (5.49–16.40 ng cm−3) and quantification (18.30–54.67 ng cm−3), intra- and inter-day precision (2.41–9.78% and 1.27—14.5%, respectively), trueness (− 8.4 to − 2.0%), and recovery (70.02–113.5%) and repeatability (2.22–10.58%) for solid-phase extraction stage were assessed. The method was evaluated using RGB additive colour model regarding the analytical and practical effectiveness and greenness of the method (achieving a brilliance score of 67.1%). Moreover, the developed method was successfully applied to determine three oligosaccharides (DSLNT, 3’SL, and 6’SL) in colostrum samples. Considering its effectiveness, the method has promise for this type of application.Graphical abstract

Design and fabrication of fluorous monoliths with tunable surface property for capillary liquid chromatography

Hierarchically porous monoliths with satisfactory properties have been employed in diverse fields, especially separation. In this study, pentafluorophenyl acrylate (PFPA), pentaerythritol tetraacrylate (PETA) and trimethylolpropane tris(3-mercaptopropionate) (TTMP) were selected as precursors to fabricate a novel monolithic column by thermally initiated polymerization in the presence of a binary porogenic system containing tetrahydrofuran and 1-propanol. The fabricated poly(PFPA-co-PETA-co-TTMP) monolithic column revealed excellent permeability and mechanical stability. Additionally, baseline separation of the mixture of small molecules can be achieved, involving alkylbenzene and fluorobenzene in chromatographic assessment, and the theoretical plate number is up to 60,500 plates/m for butylbenzene with a linear velocity of 0.14 mm/s. Tryptic digest of HeLa as an analyte was used to investigate the possibility of the poly(PFPA-co-PETA-co-TTMP) monolith in biological separation by cLC-MS/MS. Moreover, benefiting from the existence of pentafluorophenyl groups, the cucurbit[8]uril (CB[8]) could be modified on the prepared poly(PFPA-co-PETA-co-TTMP) monolith through host-guest interaction to obtain poly(PFPA-co-PETA-co-TTMP)-CB[8] monolith. It could be observed that significant changes in retention behavior of analytes appeared after immobilizing CB[8] on the monolith. It offered an innovative approach by utilizing host-guest interaction to fabricate monolithic columns with different chromatographic behaviors.

Construction of adamantane-based monolithic column with three-dimensionally porous structure for small molecules separation and biosample analysis

BackgroundThe fabrication technique of capillary column is the key to the development and application of capillary liquid chromatography (cLC) to improve separation efficiency for analytes. The capillary monolithic column possessed three-dimensionally connected porous or channel structures. Unique porous structure endows excellent permeability and high performance in diverse fields, especially in separation. Thereinto, organic monolithic columns have attracted widespread attention due to their advantages of simple preparation and excellent biocompatibility. However, their separation selectivity needs to be further developed and regulated to apply the separation of more diverse samples. ResultsA novel polymeric monolithic column was prepared via thermally initiated in situ copolymerization of 2-methyladamantan-2-yl acrylate (MADA) with ditrimethylolpropane tetraacrylate (DTTA) in fused silica. The prepared poly(MADA-co-DTTA) monolith showed adjustable permeability, developed porous structure and high thermal stability. Consequently, it exhibited excellent separation capability of small molecules (alkylbenzenes and polycyclic aromatic hydrocarbons). Especially, when acetonitrile/water (60/40, v/v) was used as the mobile phase, the theoretical plate numbers reached 84,000 plates m−1 for butylbenzene at a linear velocity of 0.5 mm s−1. Most importantly, the hydrophobicity of the poly(MADA-co-DTTA) monolithic column was regulated via host-guest interaction between adamantyl group and cucurbit [7]uril (CB[7]). Additionally, the poly(MADA-co-DTTA) monolith was further adopted for the analysis of the tryptic digest of proteins from HeLa by cLC-MS/MS. The 33,783 unique peptides and 5,299 proteins were identified on the monolith, which exhibited great separation ability for complex samples. Significance and noveltyDue to abundant pore structure and good chemical properties, the poly(MADA-co-DTTA) monolithic column exhibited high performance for the separations of small molecules and biological sample. Meanwhile, owing to the existence of adamantyl-group, CB[7] was immobilized on the poly(MADA-co-DTTA) monolithic column to fabricate poly(MADA-co-DTTA)-CB[7] by host-guest interaction. It is possible to adjust the surface chemistry of the monolithic materials to accommodate more complex analytes.

Analysis of Single Sub-Millimeter Microplastic Particles by Capillary Size Exclusion Chromatography

Analysis of Single Sub-Millimeter Microplastic Particles by Capillary Size Exclusion Chromatography

Intact quantitation of cysteine-conjugated antibody-drug conjugates using native mass spectrometry.

A common strategy for antibody-drug conjugate (ADC) quantitation from in vivo study samples involves measurement of total antibody, conjugated ADC, and free payload concentrations using multiple reaction monitoring (MRM) mass spectrometry. This not only provides a limited picture of biotransformation but can also involve lengthy method development. Quantitation of ADCs directly at the intact protein level in native conditions using high-resolution mass spectrometers presents the advantage of measuring exposure readout as well as monitoring the change in average drug-to-antibody ratio (DAR) and in vivo stability of new linker payloads with minimal method development. Furthermore, site-specific cysteine-conjugated ADCs often rely on non-covalent association to retain their quaternary structure, which highlights the unique capabilities of native mass spectrometry (nMS) for intact ADC quantitation. We developed an intact quantitation workflow involving three stages: automated affinity purification, nMS analysis, and data processing in batch fashion. The sample preparation method was modified to include only volatile ion-pairing reagents in the buffer systems. A capillary size-exclusion chromatography (SEC) column was coupled to a quadrupole time-of-flight high-resolution mass spectrometer for high-throughput nMS analysis. Samples from two mouse pharmacokinetic (PK) studies were analyzed using both intact quantitation workflow and the conventional MRM-based approach. A linear dynamic range of 5-100 μg/mL was achieved using 20 μL of serum sample volume. The results of mouse in vivo PK measurement using the intact quantitation workflow and the MRM-based approach were compared, revealing excellent method agreement. We demonstrated the feasibility of utilizing nMS for the quantitation of ADCs at the intact protein level in preclinical PK studies. Our results indicate that this intact quantitation workflow can serve as an alternative generic method for high-throughput analysis, enabling an in-depth understanding of ADC stability and safety in vivo.

Abstract PO1-13-08: Comparative analysis of stearoyl-CoA desaturase 1 expression and activity index in model cell lines of breast cancer subclasses

Abstract Introduction Due to the unique conditions of the tumor microenvironment, such as hypoxia, poor angiogenesis, and mitochondrial dysfunction, cancer cells face challenges in obtaining nutrients and energy supply. However, monounsaturated fatty acids have been identified as preferred substrates for energy generation and maintenance of cellular structures in cancer cells. These fatty acids can be obtained through de novo fatty acid synthesis. Stearoyl-CoA desaturase 1 (SCD1) is the primary enzyme involved in the biosynthesis of monounsaturated fatty acids from saturated fatty acids and is known to be upregulated in various malignancies, including breast cancer. This study aimed to assess the expression and activity index of SCD1 in well-characterized MCF-7, SK-BR3, and MDA-MB231 breast cancer cell lines, representing luminal A/B, HER2-enriched, and triple-negative subtypes, respectively. Methods The breast cancer cell lines were cultured in RPMI-1640 medium with 10% fetal bovine serum at 37°C, 5% CO2, and 95% humidity for 72 hours. After harvesting the cells, SCD1 expression was analyzed using real-time quantitative PCR, and the activity index was determined by measuring the ratio of monounsaturated fatty acids to saturated fatty acids using capillary gas-liquid chromatography. Results MCF-7 cell line showed a significantly higher SCD1 expression compared to MDA-MB231 (250-fold, p< 0.0001) and SK-BR3 (83-fold, p< 0.0001) cell lines. The activity index of SCD1 was significantly higher in both MCF-7 and MDA-MB231 cell lines with fold changes of 2.6 and 2.5, respectively, compared to SK-BR3 cell line (p=0.004 for both). Conclusion Cell model assays demonstrated that the luminal A/B and triple-negative subtypes showed a higher capacity to convert saturated fatty acids to monounsaturated fatty acids through SCD1 enzyme activity, indicating a different lipid metabolism profile in the HER2-enriched subtype. These new findings reveal differences in SCD1 expression and activity among breast cancer subclasses. They highlight vulnerabilities in metabolism and identify potential therapeutic targets with clinical implications for managing breast cancer. Citation Format: Hamid-Reza Feizi, Amir Mehdizadeh, Zohreh Sanaat, Khadijeh Abbasi, Frank Gieseler, Masoud Darabi. Comparative analysis of stearoyl-CoA desaturase 1 expression and activity index in model cell lines of breast cancer subclasses [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO1-13-08.

Professor Carla Bottoli, an enthusiastic scientific researcher, kindly spoke to BrJAC

Carla Beatriz Grespan Bottoli holds a degree in Industrial Chemistry from the Federal University of Santa Maria (1996), as well as a Ph.D. in Chemistry (2002), and a postdoctoral degree in Chemistry (2002-2004), both from the State University of Campinas (Unicamp). In 2005, she undertook an internship at the Institute of Nuclear Sciences of the National Autonomous University of Mexico (UNAM) to study radiolysis in vitamins using gamma radiation from a source of Cobalt 60. In 2017, she presented her habilitation thesis (associate professor's thesis) at Unicamp. Currently, she is Associate Professor and Postgraduate Advisor at the Institute of Chemistry at Unicamp (IQ-Unicamp), where she works in the area of analytical chemistry, with emphasis on separation methods, mainly liquid chromatography. Prof. Bottoli coordinates the NovaCrom – Liquid Chromatography Laboratory (https://novacromlab.wixsite.com/novacrom), which is a research group that has been working on the development of new monolithic stationary phases for capillary liquid chromatography. Research is also focused on the development of analytical methods that employ liquid chromatography coupled with mass spectrometry to study the translocation of pesticides in plants such as coconut, palm, and soybean. Between 2019 and 2021, Prof. Bottoli was an associate coordinator of the Research Commission of the IQ-Unicamp and is currently a member of the Research Integrity Commission at Unicamp. She is a member of the Brazilian Chemical Society and a researcher at the National Institute of Science and Technology in Bioanalytics (INCTBio). Carla Bottoli is the mother of Murilo, 13 and Mateus, 10.

Histidine tag modified magnetic beads for analysis of arsenic binding proteins

BackgroundMany proteins with thiol groups can bind with trivalent arsenic which are termed as arsenic binding proteins, thus change their physiological functions. Therefore, it is vital to analyze the arsenic binding proteins in cells. The Pull-Down strategy based on biotinylated phenylarsenic acid (Bio-PAO(III)) probes is an effective way for analysis of arsenic binding proteins. In this strategy, streptavidin magnetic beads (SA-MBs) was applied to capture the arsenic binding proteins conjugating with Bio-PAO(III) probe. However, strong interaction between SA and biotin makes the elution of arsenic binding proteins not easy. ResultsWe developed a novel affinity separation strategy to address the challenge of eluting arsenic binding proteins, a key issue with the existing Bio-PAO(III) Pull-Down method. By employing magnetic beads modified with Nα-Bis(carboxymethyl)-l-lysine (NTA-Lys), polyhistidine-tag (His6-Tag), and SA (MB-NTA(Ni)-His6-SA), we established a more efficient purification process. This innovative approach enables selective capture of arsenic binding proteins in HepG2 cells labeled by Bio-PAO(III) probes, facilitating gentle digestion by trypsin for precise identification through capillary high performance liquid chromatography (Cap HPLC)-electrospray ionization (ESI)-tandem mass spectrometry (MS/MS). What is more, the magnetic beads can be regenerated by using imidazole as the eluent, and the obtained MB-NTA(Ni) can be reloaded with His6-SA for next use. Our method successfully identified 41 arsenic binding proteins, including those involved in cytoskeletal structure, heat shock response, transcriptional regulation, DNA damage repair, redox state regulation, mitochondrial dehydrogenase function, and protein synthesis and structure. SignificanceThis work contributes to a more comprehensive understanding of the toxic mechanisms of arsenic, potentially providing valuable insights for the prevention or treatment of arsenic-related diseases.

High-Throughput Capillary Liquid Chromatography Using a Droplet Injection and Application to Reaction Screening.

The cycle time of a standard liquid chromatography (LC) system is the sum of the time for the chromatographic run and the autosampler injection sequence. Although LC separation times in the 1-10 s range have been demonstrated, injection sequences are commonly >15 s, limiting throughput possible with LC separations. Further, such separations are performed on relatively large bore columns requiring flow rates of ≥5 mL/min, thus generating large volumes of mobile phase waste when used for large scale screening and increasing the difficulty in interfacing to mass spectrometry. Here, a droplet injector system was established that replaces the autosampler with a four-port, two-position valve equipped with a 20 nL internal loop interfaced to a syringe pump and a three-axis positioner to withdraw sample droplets from a well plate. In the system, sample and immiscible fluid are pulled alternately from a well plate into a capillary and then through the injection valve. The valve is actuated when sample fills the loop to allow sequential injection of samples at high throughput. Capillary LC columns with 300 μm inner diameter were used to reduce the consumption of mobile phase and sample. The system achieved 96 separations of 20 nL droplet samples containing 3 components in as little as 8.1 min with 5-s cycle time. This system was coupled to a mass spectrometer through an electrospray ionization source for high-throughput chemical reaction screening.