LC System Research Articles

LC Headings and Classification from May-June 2024 Lists

LC Headings and Classification from May-June 2024 Lists

Comprehensive chromatographic profiling and structural analysis of key anticoagulant components in enoxaparin

Heparin is the most widely used anticoagulant in clinical practice, with enoxaparin being one of the most important low molecular weight heparins (LMWHs). In this study, an antithrombin III (ATIII) affinity column was used. Enoxaparin and its oligosaccharides of varying sizes, prepared using preparative size exclusion chromatography (SEC), were fractionated through the ATIII affinity column. The different affinity fractions from each oligosaccharide size were profiled using strong anion exchange (SAX) chromatography. Each peak was automatically transferred to an SEC column for desalting prior to mass spectrometry (MS) analysis, which enabled structural identification using a multiple heart-cut (MHC) 2D LC-MS system (SAX-SEC-MS). The high-affinity fraction from enoxaparin was further analyzed using the MHC 2D LC system (SEC-SAX). SAX profiles of the high-affinity oligosaccharides, prepared by both size and affinity fractionation, were consistent with those obtained by direct SEC-SAX analysis. The possible sequences of several high-affinity hexasaccharides and the domain compositions of high-affinity octa- and decasaccharides in enoxaparin were further elucidated by disaccharide analysis after manual collection of the oligosaccharides. This work advances the understanding of enoxaparin's structural features and offers a potential approach to improve the quality of enoxaparin, as well as to identify key structural motifs in heparin/LMWHs that contribute to protein binding.

B-150 Chromatographic Separation and Quantitation of Human Insulin and Lispro Analog in Serum Using Liquid Chromatography High-Resolution Mass Spectrometry

Abstract Background Factitious hypoglycemia, a deliberate attempt to induce low blood glucose levels occurring secondary to the surreptitious administration of exogenous insulin, can be difficult to diagnose due to a variety of factors including the increased use of synthetic insulin analogs in clinical practice. Traditional insulin immunoassays demonstrate variable cross-reactivity with commonly prescribed insulin analogues and their metabolites, making it difficult to measure specific analogs. The development of liquid chromatography high resolution accurate mass (LC-HRAM)-mass spectrometry (MS) assays has largely circumvented the limitations of traditional immunoassays for detection and quantitation of insulin analogs. Analytical challenges remain, however, specifically in differentiating lispro, an isomeric human insulin analog formed by the transposition of proline and lysine near the C-terminal end of the B chain, from human insulin in patient samples due to the equivalent mass-to-charge ratios of their precursor ions. Methods Immunoaffinity purification of insulin from calibrators and quality control material prepared by spiking insulin-depleted serum with pharmaceutical insulin preparations, as well as patient samples, was performed using tips coupled with anti-insulin monoclonal antibody. An automated liquid handler was programmed to perform aspiration and dispense cycles. Eluate from the purification protocol was submitted for analysis using a TLX-2 multiplex LC system paired with a Thermo Scientific Q Exactive Plus mass spectrometer. Various HPLC column chemistries were evaluated, a column passivation procedure was established, and column heater temperature was optimized to identify a combination of parameters yielding the best chromatographic separation of human insulin and lispro. Additionally, parallel reaction monitoring (PRM) targeted tandem mass spectrometry (MS/MS) analysis was utilized in combination with the full scan data to increase specificity through the monitoring of fragment ions produced from human insulin and each insulin analog. Results We found that a 1000 Å Diphenyl, 2.7 µm, 2.1 x 100 mm analytical column in a column heater maintained at a temperature of 80°C allowed for human insulin and lispro to be differentiated as two chromatographically separated extracted ion chromatographic peaks (XICs) and quantified down to 2.5 mcIU/mL with near-baseline separation. Before use, the column required passivation with a high protein buffer (0.05% bovine serum albumin in 20% acetonitrile [ACN] + 0.2% acetic acid) followed by increasingly organic buffers (20% ACN + 0.2% acetic acid and 50% ACN + 0.2% acetic acid). MS/MS was used to confirm the presence of analogs detected. Conclusions The LC-HRAM-MS method described here, coupled with optimized column chemistry, column passivation, and proper column temperature control allows for chromatographic separation of human insulin and lispro. Incorporating a PRM scan into the mass spectrometry full scan method provides confirmation of the insulin detected in the XICs. The results obtained from preliminary studies suggest this method is appropriate for use to facilitate the clinical investigation of suspected factitious hypoglycemia with the ability to differentiate isomeric compounds, such as human insulin and lispro.

Determination of adalimumab by HPLC with fluorescence detection using the König reaction

Monoclonal antibody drugs (mAb) are widely used to treat various diseases. Keeping quality of mAbs is crucial to maximize efficacy and minimize adverse effects. To this end, mAb content in the drug product must be precisely quantified. As methods for the determination of mAbs, absorbance measurement at 280 nm, the ligand binding assay based on ELISA, or the LC-MS/MS method based on the surrogate peptide approach is usually used. However, these methods have drawbacks in terms of selectivity, reliability, and cost, respectively. The present method is based on the surrogate peptide approach where a peptide specific to the mAb is determined by a post-column derivatization LC system with fluorescence detection and the König reaction, which is usually used for the detection of cyanide and its derivatives. The detection mechanism was the production of cyanide from the surrogate peptide by chloramine T and the generated cyanide was detected by the König reaction. In this study, adalimumab was used as a model target protein. The calculated LOD and LOQ of the present method were 0.31 μg/mL and 0.94 μg/mL, respectively. The intra-day accuracies and precisions were 127.1 % and 2.94 % for 5 μg of adalimumab and 101.6 % and 10.2 % for 50 μg of adalimumab, respectively. The inter-day accuracies and precisions were 121.8 % and 5.71 % for 5 μg of adalimumab and 101.8 % and 6.98 % for 50 μg of adalimumab, respectively. Our quantitatively determined amount of adalimumab in Humira® was in good agreement with the specified value. This method only requires a conventional LC system, not an LC-MS/MS system. By extending the application of the König reaction, we open the door to a new surrogate peptide approach for the determination of proteins including monoclonal antibody drugs.

Chemical Profiling, In Silico Pharmacokinetics Analysis and Molecular Docking Study of Phytocompounds of Homeopathy Medicine Echinacea angustifolia Q against Suppurative Bacterial Proteins

Background Computer-aided drug design is the emerging field for identification of drug action on its target organs. The pharmacokinetic study helps find out drug ADMET (absorption, distribution, metabolism, excretion, and toxicity) and drug likeness properties. PyRx software helps with molecular docking of the bioactive compounds on their target organ within a short period of time. In homeopathy, Echinacea angustifolia is one of the most commonly used drugs for microbial infective skin lesions used in internal administration as well as an external application. Objective This study was aimed at identifying the compounds present in Echinacea angustifolia Q through liquid chromatography mass spectrometry (LC-MS), ADMET, and drug likeness properties, followed by molecular docking by PyRx software to recognize the interaction between drug compounds against 12 target microbial proteins of commonly infective suppurative bacteria. Materials and Methods An analysis of the phytoconstituents of Echinacea angustifolia Q was performed using the Agilent 1290 Infinity LC system coupled to the Agilent 6545 Accurate Mass Quadrupole Time-of-Flight (QTOF) with Agilent Jet Stream Thermal Gradient Technology. In silico ADMET and drug likeness properties were assessed by free online tools, followed by a detailed docking study by PyRx software. The drug compounds with the lower glide docking score are considered to have the best binding affinity. Result The five compounds of Echinacea angustifolia Q satisfy all properties of ADMET and drug likeness. The three compounds of Echinacea angustifolia Q have low glide score of >-7.0 kcal/mol against the three microbial target proteins of Salmonella enterica, Proteus mirabilis, and Staphylococcus aureus. Conclusion Echinacea angustifolia Q has fulfilled the ADMET and drug-likeness properties for internal administration as well as external application for various suppurative bacterial infective disorders, which were confirmed through molecular docking. Therefore, Echinacea angustifolia Q may function as a potential therapeutic agent for the treatment of illnesses associated with antimicrobial resistance.

Theoretical predictions to facilitate the method development in slalom chromatography for the separation of large DNA molecules

Slalom chromatography (SC) re-emerged in 2024 due to the availability of low adsorption ultra-high pressure liquid chromatography (UHPLC) packed columns/instruments and large modalities being investigated in the context of cell and gene therapies. The physico-chemical principles of SC retention combined with hydrodynamic chromatography (HDC) exclusion have been recently reported. In SC, DNA macromolecules are retarded because: (1) they can be stretched to lengths comparable to the particle diameter, and (2) their elastic relaxation time is long enough to maintain them in non-equilibrium extended conformations under regular UHPLC shear flow conditions. Here, a quantitative HDC-SC retention model is consolidated. A general plate height model accounting for the band broadening of long DNA biopolymers along packed beds is also derived for supporting method development and predicting speed-resolution performance in SC.For illustration, the chromatographic speed-resolution properties in SC are predicted for the separation of specific critical pairs (4.0/4.5, 10/11, and 25/27 kbp) of linear dsDNA polymers. The calculations are performed for two available custom-made particle sizes, dp= 1.7 and 2.5μm, at a constant pressure of 10,000 psi. The predictions are directly validated from experimental data acquired using low adsorption MaxPeakTM 4.6 mm i.d. Columns packed with 1.7μm BEHTM 45 Å (15 cm long column) and 2.5μm BEH 125 Å (30 cm long column) Particles, and by injecting six linear dsDNAs (λ DNA-Hind III Digest). The LC system is very low dispersion ACQUITYTM UPLCTM I-class PLUS System, and the mobile phase is a 100 mM phosphate buffer at pH 8. Maximum resolution is always achieved when the average extended lengths of linear dsDNAs are equal to a critical length, which is proportional to the particle diameter and to the square root of the applied shear rate. Most advantageously, the experimental results reveal that the relaxation times of linear dsDNAs observed under shear flow conditions are two orders of magnitude shorter than those expected in the absence of flow: this enables the detection of the longest linear dsDNAs up to 25 kbp without irremediable loss in column performance. Finally, the retention-efficiency model elaborated in this work can be used to rapidly anticipate and develop methods (selection of particle size, column length, and operating pressure) for any targeted DNA and time-resolution constraints.

Are light curve classification metrics good proxies for SN Ia cosmological constraining power?

When selecting a lc classifier for use as part of a photometric supernova Ia (SN Ia) cosmological analysis, it is common to make decisions based on metrics of classification performance, such as the contamination within the photometrically classified SN Ia sample, rather than a measure of cosmological constraining power. If the former is an appropriate proxy for the latter, this practice would save those designing an analysis pipeline from eliminate the computational expense of a full cosmology forecast in the analysis pipeline design process This study tests the assumption that lc classification metrics are an appropriate proxy for cosmology metrics. We emulated photometric SN Ia cosmology lc samples with controlled contamination rates of individual contaminant classes and evaluated each of them under a set of classification metrics. We then derived cosmological parameter constraints from all samples under two common analysis approaches and quantified the impact of contamination by each contaminant class on the resulting cosmological parameter estimates. We observe that cosmology metrics are sensitive to both the contamination rate and the class of the contaminating population, whereas the classification metrics are shown to be insensitive to the latter. Based on these findings, we discourage any exclusive reliance on classification-based metrics for analysis design decisions, which (counterintuitively) include but are not limited to the classifier choice. Instead, we recommend optimising science analysis pipeline design choices using a metric of the information gained about the physical parameters of interest.

Development and clinical implementation of an LC-HRMS method for ivacaftor, lumacaftor, tezacaftor and elexacaftor in human plasma and breast milk.

The four cystic fibrosis (CF) transmembrane conductance regulator (CFTR) modulators, ivacaftor, lumacaftor, tezacaftor, and elexacaftor, have revolutionised the treatment of CF by direct action on the protein target behind the disease's development. The aim was to develop and validate a quantification method for these CFTR modulators in plasma and breast milk to better understand inter-patient variability in pharmacokinetics and treatment outcome, including the risk of adverse drug reactions. The ability to monitor CFTR modulators in breast milk enables the estimation of the exposure of breastfed infant, with a potential concern for CFTR modulator-induced liver injury. The analysis was performed on a Thermo Vanquish Flex Binary UHPLC system coupled to a high-resolution mass spectrometer (HRMS), Thermo Q Exactive. The analytes were detected using positive electrospray ionisation in full scan mode. After sample preparation by protein precipitation, the supernatant was injected onto the LC system and the analytes were separated using a Zorbax SB-C18 Rapid Res HPLC column (3.5µm, 4.6 × 75mm). This is the first published method for CFTR modulators in breast milk. The validated quantification range for ivacaftor is 0.0050-10µg/mL with a coefficient of variation < 6% and a mean accuracy of 97-106%; for lumacaftor, tezacaftor, and elexacaftor, the validated quantification range is 0.050-100µg/mL with a coefficient of variation < 8% and a mean accuracy 93-106%. A simple and sensitive quantification method for CFTR modulators has been developed and used for routine analysis of human plasma and breast milk samples since 2022.

Targeted screening and profiling of massive components of colistimethate sodium by two-dimensional-liquid chromatography-mass spectrometry based on self-constructed compound database

In-depth study of the components of polymyxins is the key to controlling the quality of this class of antibiotics. Similarities and variations of components present significant analytical challenges. A two-dimensional (2D) liquid chromatography-mass spectrometric (LC-MS) method was established for screening and comprehensive profiling of compositions of the antibiotic colistimethate sodium (CMS). A high concentration of phosphate buffer mobile phase was used in the first-dimensional LC system to get the components well separated. For efficient and high-accuracy screening of CMS, a targeted method based on a self-constructed high resolution mass spectrum database of CMS components was established. The database was built based on the commercial MassHunter Personal Compound Database and Library (PCDL) software and its accuracy of the compound matching result was verified with 6 known components before being applied to genuine sample screening. On this basis, the unknown peaks in the CMS chromatograms were deduced and assigned. The molecular formula, group composition and origins of a total of 99 compounds, of which the combined area percentage accounted for more than 95% of CMS components, were deduced by this 2D-LC-MS method combined with the MassHunter PCDL. This profiling method was highly efficient and could distinguish hundreds of components within 3 h, providing reliable results for quality control of this kind of complex drugs.

Recent advances and applications in drug analysis by nano-scale separation techniques

Nano-scale separation techniques (NSTs) offer significant advantages in relation to drug analysis in a wide range of samples. NSTs, including low flow rate LC systems or capillary or chip based electrophoresis/electrochromatography systems, have become the primary tool for advanced drug analysis, and indispensable technology for sensitive and selective drug analysis. In recent decades, significant advances have been achieved using NSTs for drug analysis. In this review, sample preparation strategies, new advances and applications in NSTs and the contribution toward forensic science applications were reported. In addition, some recent and selected applications with or without mass spectrometry (e.g., low resolution/high resolution -MS) are summarized.

Validation of Commercial Formulation of Difenoconazole Using HPLC Equipped with Dad

Effective, selective, precise, and accurate liquid chromatographic analytical methods for the determination of difenoconazole fungicide in the formulation have been optimized and validated to meet the accreditation requirements, the performance characteristics of the analytical method were validated which is also one of the basic requirements of ISO Standard 17025:2017, for the rapid determination of difenoconazole by HPLC-DAD. The used method involves the extraction of the substances by sonication of the sample with acetonitrile, followed by dilution in acetonitrile, and direct injection on a liquid chromatography system. For the analysis LC system from Agilent Technologies 1290 Infinity II was used. Good separation was achieved on a Luna C18 column (3µm, 100°A, 3x150mm) with a guard column (C18 4x2.0mm I.D), an isocratic mobile elution consisting of acetonitrile and water acidified 0.075% with formic acid (85:15, v/v), at a flow rate of 0.7 ml/minute and UV detection at 220 nm. The column temperature was 25⁰C, injected volume was 1 μL. The analysis duration was 10 minutes (the retention time of difenoconazole and 4 – Hydroxybenzoic acid – methyl ester was 1.846 and 1.135 minutes, respectively. The linearity within the concentration range of 750-125 µg mL-1, with the internal standard at a concentration of 250 µg mL-1 with an average correlation coefficient (R2) of 0.9998. We have considered precision, repeatability, and selectivity in the validation.

High-Throughput Proteomics Enabled by a Fully Automated Dual-Trap and Dual-Column LC-MS.

This Technical Note describes a dual-column liquid chromatography system coupled to mass spectrometry (LC-MS) for high-throughput bottom-up proteomic analysis. This system made full use of two 2-position 10-port valves and a binary pump with an integrated loading pump of a commercial LC instrument to provide successive operation of two parallel subsystems. Each subsystem consisted of a set of trap columns and an analytical column. A T-junction union was used to split the mobile phase from the loading pump into two parts. This allowed one set of columns to be washed and equilibrated, followed by the injection of the next sample, while the previous sample was eluting and being analyzed on the other set of columns, thereby greatly increasing the analysis throughput. This approach showed high reproducibility for the analysis of HeLa tryptic digests with average relative standard deviation (RSD) values of 1.75%, 6.90%, and 5.19% for the identification number of proteins, peptides, and peptide-spectrum matches (PSMs), respectively, across 10 consecutive runs. The capacity for peptide and protein identification, as well as proteome depth, of the dual-column LC system was comparable to a conventional single-column system. Due to its simple equipment requirements and set up process, this method should be highly accessible for other laboratories.

Liquid chromatography and liquid chromatography coupled with tandem mass spectrometry studies for the identification and characterization of degradation products of lobeglitazone

AbstractThe objective of the present research work was to demonstrate the application of liquid chromatography coupled with tandem mass spectrometry (LC‐MS/MS) for the separation, identification, and characterization of degradation products (DPs) of the drug lobeglitazone. The drug was subjected to various stress conditions such as oxidation, hydrolysis, thermal, and photolytic degradation as per the guidelines of the International Conference on Harmonization Q1A (R2). The drug was stable under thermal stress conditions, whereas it was susceptible to degradation in the other stress conditions forming four DPs. DPs were separated using a high‐performance LC system equipped with Zorbax SB C18 column (250 × 4.6 mm, 5 µm particles) using isocratic elution mode. The DPs were identified and characterized using MS and MS/MS. The chemical structure and fragmentation pattern were predicted for each degradant from the data obtained by mass studies. The drug and identified DPs were assessed by in‐silico approaches for predicting absorption, distribution, metabolism, and toxicity behavior. The in‐silico tools used for prediction were the pkCSM web server, ToxTree, and OSIRIS property explorer.

Thermal performance analysis of battery thermal management system utilizing bionic liquid cooling plates with differentiated velocity distribution strategy

Bionic channel liquid cooling plates (BC-LCPs) show high heat dissipation performance in battery thermal management system (BTMS), but suffer from the poor temperature-equalizing ability. Herein, a BTMS with novel BC-LCPs and differentiated velocity distribution strategy is proposed to overcome the low temperature uniformity, and balance the cooling performance and power consumption. The BC-LCPs are firstly designed by precisely tailoring the series cobweb channels inspired by the bionic structures in the nature. Through systematic optimization combining single factor analysis with orthogonal test and complementary test, differentiated velocities (vI and vII) of 0.20 and 0.46 m∙s−1 are adopted, respectively. Ultimately, the LC system demonstrates exceptional cooling performance while maintaining lower energy consumption. For example, with a discharge rate of 3C and an environment temperature of 50 °C, the temperature and temperature difference of the battery pack are controlled synergistically below 30.9 and 4.47 °C, respectively. At the same time, the Ptotal can reach as low as 0.202 W, which is reduced by 42.0 % compared to the case with uniform velocity strategy.

Abstract PO5-09-02: Breast Tissue Proteomic Profile of Breast Cancer in Premenopausal Women and Association with Mammographic Breast Density

Abstract Introduction: Breast cancer incidence is rising in premenopausal, hence, there is a critical need to understand factors underlying premenopausal breast cancer development in order to guide targeted prevention. Mammographic breast density is a strong risk factor for, as well as an intermediate phenotype for premenopausal breast cancer. Yet, the molecular mechanisms underlying the associations of dense breasts with breast cancer are not well understood. Our objectives in this study are to perform proteomic analysis in breast tissues to (i) identify proteins that are associated with breast cancer development in premenopausal women; (ii) determine which of these proteins are also associated with dense breasts. Methods: We performed proteomic analysis on tumor and adjacent normal tissues from 50 premenopausal women with breast cancer who had breast tissue samples archived at the St. Louis Breast Tumor Registry. Samples were analyzed on Orbitrap Eclipse Tribrid coupled with Vanquish Neo LC system (Thermo Fisher Scientific). Database search was performed using MaxQuant and MS1 LFQ intensities were used for further data analysis. We performed multiple imputation using Bayesian Principal Component Analysis and hierarchical clustering analysis using ‘ConsensusClusterPlus’ package in R. Pathway enrichment analysis was performed using Reactome Pathway database. 10 times repeated 5-fold cross validation using Logistic Regression (LR) was performed for model development. Top 10 proteins were selected by variable importance in logistic regression, and recursive feature elimination was performed for feature selection. We corrected for multiple testing by setting the false discovery rate, FDR, p-value &amp;lt; 0.05. Results: After imputing for missing values, we identified 3,571 proteins that were expressed in tumor and normal breast tissues. We selected 1,640 proteins for further analysis using median absolute deviation analysis. In multivariable-adjusted analysis, 451 proteins were up-regulated, and 180 proteins were down-regulated in tumor tissues compared to adjacent normal tissues (FDR &amp;lt; 0.05). 53 of the 451 proteins that were upregulated in tumor tissues were also upregulated in dense breasts. Some of the top proteins that were upregulated in both tumor tissues and dense breasts were AIMP2, ALDH18A1, BZW1, CKAP5, COPG1, ERGIC1, GSPT1, ILF2, IPO7, LRBA, and PSMD12. Mammographic breast density protein clusters were highly correlated with hormone receptor positivity clusters and were enriched in ESR-mediated signaling, IGF1R signaling, NOTCH4, PTEN regulation, and NFkB pathway. Discussion: Using untargeted proteomics, we identified proteomic signatures of breast cancer development in premenopausal women. We further identified protein biomarkers and pathways that are shared between breast tumors and dense breast tissues in premenopausal women. Our novel findings highlight mammographic breast density as a strong intermediate phenotype that can be targeted in breast cancer prevention in premenopausal women. Larger studies are needed to validate our findings. Citation Format: Shaili Tapiavala, Minsoo Son, Graham Colditz, Ah Young Goo, Adetunji Toriola. Breast Tissue Proteomic Profile of Breast Cancer in Premenopausal Women and Association with Mammographic Breast Density [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 PO5-09-02.

Treat It Like a Circuit, Part II: Applications and Troubleshooting

The analogy that electrons flowing in wires is like water flowing through a tube can be remarkably effective for teaching and learning about fluid flow in LC systems. In this installment, we apply the concepts developed in last month’s installment by demonstrating how they can be used to help troubleshoot problems in LC involving pressure and flow. We also introduce several free tools that can be used to calculate pressure drops in different elements of LC systems, including connecting capillaries and packed columns. Knowing what pressure drops to expect for system components under different chromatographic conditions is very valuable in many troubleshooting situations.

LC Headings and Classification from September 2023 – February 2024 Lists

LC Headings and Classification from September 2023 – February 2024 Lists

Improved lipid analysis using a 2D-LC-MS system with a novel injection procedure

The aim of this study was to improve analysis of nonpolar lipidomics sample extracts using reversed phase (RP) chromatography. A 4/3/3 (v/v/v) mixture of methanol/methyl tert-butyl ether/chloroform (MeOH/MTBE/CHCl3, MMC) was chosen for sample extraction solvent based on its proven extraction capability for several lipid classes. To avoid carry over, loss of analytes and peak distortion the loops and all capillaries of the presented LC system were flushed and filled up with methanol until the analytical column. The choice of methanol was due to its weak elution strength and being infinitely miscible with MMC and several other nonpolar solvents. This allowed injection of a 100 μl sample that was 20 μl nonpolar extraction solvent diluted fivefold with methanol. All lipids of 25 lipid classes were transferred quantitatively to the column head where the online dilution of methanol was carried out with aqueous eluent for focusing the lipid analytes. The weak elution strength of methanol prevented peak distortions. The consecutive reversed phase elution resulted in remarkably narrow peaks (full width at half maximum was 0.07–0.08 min typically) and enhanced sensitivity (limit of detection usually in sub nM region) because of increased sample injection volume and narrow peaks. Calibration and quality control samples made by diluting commercial lipid standards 200–50000 times confirmed the applicability of this approach both for targeted lipid quantification and for untargeted quantitative comparison of lipids from different sources.

Exploring Negative Chemical Ionization of Per- and Polyfluoroalkyl Substances via a Liquid Electron Ionization LC-MS Interface.

Per- and polyfluoroalkyl substances (PFASs) are a class of aliphatic manufactured compounds comprising fluoro-chemicals with varied functional groups and stable carbon-fluorine bonds. They are defined as "forever chemicals" due to their persistent and bioaccumulative character. These substances have been detected in various environmental samples, including water, air, soil, and human blood, posing significant health hazards. High-performance liquid chromatography coupled with electrospray ionization mass spectrometry (HPLC-ESI-MS) is typically employed for the analysis of PFASs. Negative chemical ionization (NCI) is generally coupled to gas chromatography (GC) and offers high selectivity and sensitivity for compounds containing electronegative atoms, such as PFASs. The liquid electron ionization (LEI) interface is an efficient mechanism developed to robustly couple a liquid flow rate from an LC system to an EI or a CI source. This interface has been successfully utilized for pesticide determination in UHPLC-LEI-CI in negative ion mode (NCI). This work aims to evaluate different parameters involved in the ionization of PFASs analyzed in LC-LEI-NCI and subsequently develop a method for their detection in real samples. The parameters considered for this study include (i) a comparison of different CI reagent gases (methane, isobutane, and argon); (ii) the use of acetonitrile as both the chromatographic solvent and CI reagent gas; (iii) the presence of water and formic acid as chromatographic mobile phase components; and (iv) the mobile phase flow rate. The optimal combination of these parameters led to promising results. Tentative fragmentation pathways of PFASs in NCI mode are proposed based on the dissociative electron capture mechanism.

An Automated and Fast Sample Preparation Workflow for Laser Microdissection Guided Ultrasensitive Proteomics

Spatial tissue proteomics integrating whole-slide imaging, laser microdissection, and ultrasensitive mass spectrometry is a powerful approach to link cellular phenotypes to functional proteome states in (patho)physiology. To be applicable to large patient cohorts and low sample input amounts, including single-cell applications, loss-minimized and streamlined end-to-end workflows are key. We here introduce an automated sample preparation protocol for laser microdissected samples utilizing the cellenONE robotic system, which has the capacity to process 192 samples in 3 h. Following laser microdissection collection directly into the proteoCHIP LF 48 or EVO 96 chip, our optimized protocol facilitates lysis, formalin de-crosslinking, and tryptic digest of low-input archival tissue samples. The seamless integration with the Evosep ONE LC system by centrifugation allows ‘on-the-fly’ sample clean-up, particularly pertinent for laser microdissection workflows. We validate our method in human tonsil archival tissue, where we profile proteomes of spatially-defined B-cell, T-cell, and epithelial microregions of 4000 μm2 to a depth of ∼2000 proteins and with high cell type specificity. We finally provide detailed equipment templates and experimental guidelines for broad accessibility.