On-line Liquid Chromatography Research Articles

Automated High-Throughput Biological Sex Identification from Archeological Human Dental Enamel Using Targeted Proteomics.

Biological sex is key information for archeological and forensic studies, which can be determined by proteomics. However, the lack of a standardized approach for fast and accurate sex identification currently limits the reach of proteomics applications. Here, we introduce a streamlined mass spectrometry (MS)-based workflow for the determination of biological sex using human dental enamel. Our approach builds on a minimally invasive sampling strategy by acid etching, a rapid online liquid chromatography (LC) gradient coupled to a high-resolution parallel reaction monitoring (PRM) assay allowing for a throughput of 200 samples per day (SPD) with high quantitative performance enabling confident identification of both males and females. Additionally, we developed a streamlined data analysis pipeline and integrated it into a Shiny interface for ease of use. The method was first developed and optimized using modern teeth and then validated in an independent set of deciduous teeth of known sex. Finally, the assay was successfully applied to archeological material, enabling the analysis of over 300 individuals. We demonstrate unprecedented performance and scalability, speeding up MS analysis by 10-fold compared to conventional proteomics-based sex identification methods. This work paves the way for large-scale archeological or forensic studies enabling the investigation of entire populations rather than focusing on individual high-profile specimens. Data are available via ProteomeXchange with the identifier PXD049326.

Mass spectrometry-based structure-specific N-glycoproteomics and biomedical applications.

N-linked glycosylation is a common posttranslational modification of proteins that results in macroheterogeneity of the modification site. However, unlike simpler modifications, N-glycosylation introduces an additional layer of complexity with tens of thousands of possible structures arising from various dimensions, including different monosaccharide compositions, sequence structures, linking structures, isomerism, and three-dimensional conformations. This results in additional microheterogeneity of the modification site of N-glycosylation, i.e., the same N-glycosylation site can be modified with different glycans with a certain stoichiometric ratio. N-glycosylation regulates the structure and function of N-glycoproteins in a site- and structure-specific manner, and differential expression of N-glycosylation under disease conditions needs to be characterized through site- and structure-specific quantitative analysis. Numerous advanced methods ranging from sample preparation to mass spectrum analysis have been developed to distinguish N-glycan structures. Chemical derivatization of monosaccharides, online liquid chromatography separation and ion mobility spectrometry enable the physical differentiation of samples. Tandem mass spectrometry further analyzes the macro/microheterogeneity of intact N-glycopeptides through the analysis of fragment ions. Moreover, the development of search engines and AI-based software has enhanced our understanding of the dissociation patterns of intact N-glycopeptides and the clinical significance of differentially expressed intact N-glycopeptides. With the help of these modern methods, structure-specific N-glycoproteomics has become an important tool with extensive applications in the biomedical field.

Expediting online liquid chromatography for real-time monitoring of product attributes to advance process analytical technology in downstream processing of biopharmaceuticals

The application of Process Analytical Technology (PAT) principles for manufacturing of biotherapeutics proffers the prospect of ensuring consistent product quality along with increased productivity as well as substantial cost and time savings. Although this paradigm shift from a traditional, rather rigid manufacturing model to a more scientific, risk-based approach has been advocated by health authorities for almost two decades, the practical implementation of PAT in the biopharmaceutical industry is still limited by the lack of fit-for-purpose analytical methods. In this regard, most of the proposed spectroscopic techniques are sufficiently fast but exhibit deficiencies in terms of selectivity and sensitivity, while well-established offline methods, such as (ultra-)high-performance liquid chromatography, are generally considered as too slow for this task. To address these reservations, we introduce here a novel online Liquid Chromatography (LC) setup that was specifically designed to enable real-time monitoring of critical product quality attributes during time-sensitive purification operations in downstream processing. Using this online LC solution in combination with fast, purpose-built analytical methods, sampling cycle times between 1.30 and 2.35 min were achieved, without compromising on the ability to resolve and quantify the product variants of interest. The capabilities of our approach are ultimately assessed in three case studies, involving various biotherapeutic modalities, downstream processes and analytical chromatographic separation modes. Altogether, our results highlight the expansive opportunities of online LC based applications to serve as a PAT tool for biopharmaceutical manufacturing.

Spectral averaging with outlier rejection algorithms to increase identifications in top-down proteomics.

The identification of proteoforms by top-down proteomics requires both high quality fragmentation spectra and the neutral mass of the proteoform from which the fragments derive. Intact proteoform spectra can be highly complex and may include multiple overlapping proteoforms, as well as many isotopic peaks and charge states. The resulting lower signal-to-noise ratios for intact proteins complicates downstream analyses such as deconvolution. Averaging multiple scans is a common way to improve signal-to-noise, but mass spectrometry data contains artifacts unique to it that can degrade the quality of an averaged spectra. To overcome these limitations and increase signal-to-noise, we have implemented outlier rejection algorithms to remove outlier measurements efficiently and robustly in a set of MS1 scans prior to averaging. We have implemented averaging with rejection algorithms in the open-source, freely available, proteomics search engine MetaMorpheus. Herein, we report the application of the averaging with rejection algorithms to direct injection and online liquid chromatography mass spectrometry data. Averaging with rejection algorithms demonstrated a 45% increase in the number of proteoforms detected in Jurkat T cell lysate. We show that the increase is due to improved spectral quality, particularly in regions surrounding isotopic envelopes.

Elucidating Dissolved Organic Sulfur in the Coastal Environment by Improved Online Liquid Chromatography Coupled to FT‐ICR Mass Spectrometry

AbstractDissolved organic sulfur (DOS) is an important component of dissolved organic matter (DOM) and widely exists at the land‐atmosphere interface. However, the characterization of DOS at the molecular level among different environmental samples in coastal regions has not been sufficiently focused. Here, we investigated DOS in aerosol, rainwater, and river water samples in Shanghai using an improved method–online liquid chromatography coupled to Fourier transform ion cyclotron resonance mass spectrometry (LC‐FT‐ICR MS). This system is utilized to separate DOS from complicated environmental matrices. The results showed that the average relative abundance of DOS in three different environmental media (aerosol, rainwater, and river water samples) accounted for 16.8%, 21.9% and 13.1%, respectively. The common molecules of the three mediums in coastal Shanghai contain an abundance of aromatic and aliphatic DOS of anthropogenic origin in addition to the natural sources of sea spray. Notably, results from a new structural classification scheme for DOS demonstrate that anthropogenic sulfonates and their derivatives are widely spread, although their molecular diversity in different medium was recorded due to complex environmental conditions and diverse sources.

Proteomic changes in age‐related cataract and post‐vitrectomy cataract

Aims/Purpose: To present distinct signalling pathways involved in the development of age‐related cataract (ARC) and post‐vitrectomy cataract (PVC).Methods: Three sample groups, for example, the aqueous humour, the anterior capsule and the content of the phaco cassette, were collected during cataract surgery. The samples were collected from 12 patients without diabetes mellitus (DM), older than 75 years old, and from 7 patient patients without DM, who had undergone vitrectomy for retinal detachment in the past 12 months. The samples were prepared based on Sp3 protocol (single‐pot, solid‐phase‐enhanced, sample‐preparation). The recognition and quantification of proteins was performed with liquid chromatography online with tandem mass spectrometry. Data analysis was performed using the DIA‐NN software for identification and quantification of peptides and proteins. Perseus software was used for statistical analysis and data visualization.Results: Wnt signalling pathway and oxidation–reduction pathway were significantly upregulated in the content of the phaco cassette samples of ARC, while proteasome complex and complement/coagulation cascades pathways were upregulated in those of PVC. Regulation of complement pathway was significantly upregulated in all three samples of PVC. Lipid/fatty acid oxidation and response to hydrogen peroxide pathways were upregulated in anterior capsule samples of PVC.Conclusions: Comparisons between the three sample types of ARC and PVC, revealed proteomic profiles and deregulated pathways, that clarify the pathophysiology of those types of cataract. These data sets may allow the identification of specific proteins as druggable targets against cataract.

Leaching of biochar at different stages of anaerobic digestion: Leaching behaviors, leachate chemodiversity and effects

Biochar addition is recognized as a promising way to increase the potential of anaerobic digestion. However, few studies have investigated the interaction between biochar leaching in complex, practical anaerobic digestion systems and the negative effects that biochar leachate may have on the anaerobic digestion process. The traditional measurement methods are relatively simple, but may underestimate the complexity of dissolved organic matter in biochar leachate. In this study, the leaching behaviors of biochar and the influence of biochar leachate on anaerobic digestion were studied at the molecular level by applying ultra-performance liquid chromatography online coupled with a hybrid quadrupole orbitrap mass spectrometer (UPLC Orbitrap MS/MS) test analysis and various molecular information-based analytical methods such as van Krevelen diagrams and by conducting a series of anaerobic digestion experiments. Dissolved organic matter leaching was higher in buffered saline solution and methanol than in ultrapure water and the dissolved organic matter mainly consisted of lipids and proteins. In the lactic acid solution, a large amount of dissolved organic matter was leached from the biochar, dominated by low molecular weight dissolved organic matter such as CHO and CHON, and the lower the pH, the more was leached, suggesting that lactic acid may have an inhibitory effect on anaerobic digestion. The maximum methane production amount and microbial abundance especially key methanogenic archaea such as Methanosaeta in the sample with biochar leachate addition were lower than those for the other samples. This meant that the dissolved organic matter in the biochar leachate might inhibit the growth of microorganisms and adversely affect the acetic acid methanogenesis process. The results from this study showed that a greater number of substances with more complex structures were leached out than had been detected in previous studies. When applying biochar to anaerobic digestion in the future, it is recommended that biochar is leached first to remove toxic and detrimental substances to prevent possible inhibitory effects.

Pros and Cons of Separation, Fractionation and Cleanup for Enhancement of the Quantitative Analysis of Bitumen-Derived Organics in Process-Affected Waters—A Review

Oil sands process-affected water (OSPW) contains a diverse mixture of inorganic and organic compounds. Naphthenic acids (NAs) are a subset of the organic naphthenic acid fraction compounds (NAFCs) and are a major contributor of toxicity to aquatic species. Thousands of unique chemical formulae are measured in OSPW by accurate mass spectrometry and high-resolution mass spectrometry (MS) analysis of NAFCs. As no commercial reference standard is available to cover the range of compounds present in NAFCs, quantitation may best be referred to as “semi-quantitative” and is based on the responses of one or more model compounds. Negative mode electrospray ionization (ESI-) is often used for NAFC measurement but is prone to ion suppression in complex matrices. This review discusses aspects of off-line sample preparation techniques and liquid chromatography (LC) separations to help reduce ion suppression effects and improve the comparability of both inter-laboratory and intra-laboratory results. Alternative approaches to the analytical parameters discussed include extraction solvents, salt content of samples, extraction pH, off-line sample cleanup, on-line LC chromatography, calibration standards, MS ionization modes, NAFC compound classes, MS mass resolution, and the use of internal standards.

Exploring Charge-Detection Mass Spectrometry on Chromatographic Time Scales.

Charge-detection mass spectrometry (CDMS) enables direct measurement of the charge of an ion alongside its mass-to-charge ratio. CDMS offers unique capabilities for the analysis of samples where isotopic resolution or the separation of charge states cannot be achieved, i.e., heterogeneous macromolecules or highly complex mixtures. CDMS is usually performed using static nano-electrospray ionization-based direct infusion with acquisition times in the range of several tens of minutes to hours. Whether CDMS analysis is also attainable on shorter time scales, e.g., comparable to chromatographic peak widths, has not yet been extensively investigated. In this contribution, we probed the compatibility of CDMS with online liquid chromatography interfacing. Size exclusion chromatography was coupled to CDMS for separation and mass determination of a mixture of transferrin and β-galactosidase. Molecular masses obtained were compared to results from mass spectrometry based on ion ensembles. A relationship between the number of CDMS spectra acquired and the achievable mass accuracy was established. Both proteins were found to be confidently identified using CDMS spectra obtained from a single chromatographic run when peak widths in the range of 1.4-2.5 min, translating to 140-180 spectra per protein were achieved. After demonstration of the proof of concept, the approach was tested for the characterization of the highly complex glycoprotein α-1-acid glycoprotein and the Fc-fusion protein etanercept. With chromatographic peak widths of approximately 3 min, translating to ∼200 spectra, both proteins were successfully identified, demonstrating applicability for samples of high inherent molecular complexity.

Elucidating Dissolved Organic Sulfur in the Coastal Environment by Novel Online Liquid Chromatography Coupled to FT-ICR Mass Spectrometry

Elucidating Dissolved Organic Sulfur in the Coastal Environment by Novel Online Liquid Chromatography Coupled to FT-ICR Mass Spectrometry

Removal of miconazole from water by O3, UV/H2O2 and electrochemical advanced oxidation: Real-time process monitoring and degradation pathway elucidation

The occurrence of azole drugs in the environment is of growing concern due to their ecological toxicity as well as their potential role in the emergence of drug-resistant fungal strains. In this study, the degradation of miconazole (MCZ), a persistent antifungal agent, was studied using ozonation, photodegradation and electrochemical advanced oxidation processes. The elimination of MCZ was evaluated in deionized water (DIW) as well as simulated wastewater (SWW) matrices. Under optimal conditions, the results for DIW indicate that MCZ is highly degradable by ozonation (pH 3, 41.6 mg L−1 O3 dose, 5 min reaction time) and electrochemical (pH 3, 0.05 M Na2SO4, 10 mA, 180 min reaction time) processes, resulting in 98 % and 94 % removal, respectively. The photodegradation process (2.5 mg L−1 H2O2, 24,000 J m−2 UV dose) resulted in 80 % removal. Additionally, matrix effects in SWW were observed due to the presence of scavenging species. The ozonation and electrochemical reactors were coupled with an online liquid chromatography system for automated sampling. By-products, formed due to incomplete oxidation, were elucidated by liquid chromatography in combination with high-resolution mass spectrometry (LC-HRMS). The main degradation routes include hydroxylation, dehalogenation, intramolecular cyclization and imidazole ring-opening mechanisms. Preliminary ecological toxicity estimations revealed the formation of intermediates that were less toxic than MCZ, potentially increasing the biodegradability of the processed waters. Finally, the automated process analytical technology via online sampling from the reactor can provide a diagnostic tool for real-time process monitoring during the advanced treatment of reclaimed waters.

Synthesis, Self-Assembly Properties, and Degradation Characterization of a Nonionic Photocleavable Azo-Sulfide Surfactant Family.

We report the synthesis and characterization of a new family of maltose-derived nonionic surfactants that contain a photocleavable azo-sulfide linker (mAzo). The self-assembly properties of these surfactants were investigated using surface tension measurements to determine the critical micelle concentration (CMC), dynamic light scattering (DLS) to reveal the hydrodynamic radius of their self-assemblies, and transmission electron microscopy (TEM) to elucidate the micelle morphology. Ultraviolet-visible (UV-visible) spectroscopy confirmed the rapid photodegradation of these surfactants, but surface tension measurements of the surfactant solutions before and after degradation showed unusual degradation products. The photodegradation process was further studied using online liquid chromatography coupled with mass spectrometry (LC-MS),which revealed that these surfactants can form another photo-stable surfactant post-degradation. Finally, traditionally challenging proteins from heart tissue were solubilized using the mAzo surfactants to demonstrate their potential in biological applications.

Automated centrifugation-less milk deproteinization and homogenous liquid-liquid extraction of sulfonamides for online liquid chromatography

A novel approach to the determination of sulfonamides in milk based on the Lab-In-Syringe technique is presented. The method involves automated salting-out liquid-liquid extraction of the analytes, allowing simultaneous sample deproteination without requiring centrifugation or manual sample handling. The procedural parameters, including salt type, solvent-to-sample ratio, and salt solution volume, were studied. The extracts obtained were diluted in situ and transferred to online coupled liquid chromatography for analyte separation carried out in parallel to the subsequent extraction. In this way, a sample throughput of approximately 6 h−1 was achieved. The detection limits ranged from 25 to 32 μg L−1 using a 500 μL milk sample and spectrophotometric detection. The applicability of the developed method to sample analysis was proven by recovery values ranging from 73.2% to 94.1% (86.3% on average) for milk samples of different fat content spiked at 3 μg mL−1 level. Straightforward automation of one of the most laborious preparation steps in food analysis, i.e., deproteination, was demonstrated.

Chronic Fluoxetine Treatment of Socially Isolated Rats Modulates Prefrontal Cortex Proteome

Fluoxetine (Flx) is the most commonly used antidepressant to treat major depressive disorder. However, its molecular mechanisms of action are not defined as yet. A comparative proteomic approach was used to identify proteome changes in the prefrontal cortex (PFC) cytosolic and non-synaptic mitochondria (NSM)-enriched fractions of adult male Wistar rats following chronic social isolation (CSIS), a rat model of depression, and Flx treatment in CSIS and control rats, using liquid chromatography online tandem mass spectrometry. Flx reversed CSIS-induced depressive - like behavior according to preference for sucrose and immobility in the forced swim test, indicating its antidepressant effect. Flx treatment in controls led to an increase of the expression of cytosolic proteins involved in the microtubule cytoskeleton and intracellular calcium homeostasis and of enzymes involved in bioenergetic and transmembrane transport in NSM. CSIS downregulated the cytosolic proteins involved in proteasome pathway, and glutathione antioxidative system, and upregulated the expression of enzymes participating in mitochondrial-energy metabolism and transport. The presence of cytochrome c in the cytosol may suggest compromised mitochondrial membrane integrity. Flx treatment in CSIS rats downregulated protein involved in oxidative phosphorylation, such as complex III and manganese superoxide dismutase, and upregulated vesicle-mediated transport and synaptic signaling proteins in the cytosol, and neuronal calcium-binding protein 1 in NSM. Our study identified PFC modulated proteins and affected biochemical pathways that may represent potential markers/targets underlying CSIS-induced depression and effective Flx treatment, and highlights the role of protein systems involved in NSM and various metabolic pathways potentially involved in neuronal plasticity.

In-depth characterization of nitrogen heterocycles of petroleum by liquid chromatography-energy-resolved high resolution tandem mass spectrometry

With the increased attention to processing heavy crude oils, a detailed description of chemical composition is critical for the petroleum refining industry. The current analytical technique such as ultrahigh resolution mass spectrometry has been successfully applied for the molecular level characterization of complex petroleum fractions. But the structural characterization of heavy petroleum feedstock is still a great challenge. In this study, a novel in-depth characterization method of nitrogen heterocycles (N-heterocycles) in heavy petroleum mixtures was proposed by online liquid chromatography coupled with electrospray ionization high resolution energy-resolved mass spectrometry. A series of typical basic aromatic, neutral aromatic and naphtheno-aromatic nitrogen heterocyclic model compounds were synthesized to investigate energy-resolved fragmentation behaviors in high energy collision-induced dissociation at 10–100 eV. Energy-dependent fragmentation pathways were elucidated. Notably, characteristic double bond equivalent (DBE) versus carbon number distributions of N1 ions and all CH ions were discovered, which were closely related to their core structure. Then a workflow to assign core structures of alkyl-substituted N-heterocycles in petroleum was proposed and validated. The developed method was applied to investigate the structural isomers in feed and product vacuum gas oil (VGO) fractions. Core structural differences in feed VGO and subtle structural variations between feed and product VGOs were recognized. This work can distinguish structural isomers of N-heterocycles with the subtle difference in their core structure in heavy petroleum fractions based on global energy dimensional fragmentation characteristics.

A data processing pipeline for petroleomics based on liquid chromatography-high resolution mass spectrometry

Online liquid chromatography coupled with high-resolution mass spectrometry (LC-HRMS) has attracted much attention in the molecular characterization of crude oil. Neither open access nor commercially available petroleomics tools were developed specifically to process LC-HRMS data. Here, a novel data processing pipeline was specifically designed for LC-HRMS-based petroleomics data. A customizable formula database was established deriving from the detected sample, which could avoid the interference caused by a large number of redundant molecules in a conventionally theoretical molecular database. Molecular formula candidates were assigned by the formula database using a low noise threshold, and false-positive assignments were eliminated by the chromatographic retention behaviors. Multi-dimensional information was obtained, including heteroatom class, double bond equivalent (DBE), carbon number, retention time, and MS/MS spectra. The developed method was compared with a popular petroleomics software, similar relative abundance class distribution was obtained, and much more formulas of low abundant components were uniquely extracted by the developed method. Finally, it was applied to reveal variation between feed and product oils in hydrodenitrogenation. Significantly compositional and structural differences were revealed. The developed method provides a useful pipeline for molecular data mining of petroleum samples.

Brass wires with different surface wettability used for in-tube solid-phase microextraction

Metal wires have been widely used as substrates for solid-phase microextraction (SPME) fibers instead of commonly fragile silica fibers, but complicated coating modification of their surface is required. Herein, a series of brass wires were soaked in an acidic iron trichloride solution with ultrasonication, which etched the brass surface through a redox reaction. The surface wettability of the pristine brass wire was transformed from hydrophobic to hydrophilic owing to the formation of micro/nanoscale hierarchical structures. After modification with n-octadecanethiol (ODT) and 2-naphthalenethiol (NT), respectively, both wires exhibited superhydrophobicity. Characterization of the resulting wires was conducted using SEM and EDS, and the surface wettability was measured by a contact angle goniometer using identical brass meshes. To build an in-tube SPME–high-performance liquid chromatography (HPLC) online system, the extraction tube was connected with HPLC equipment by replacing the sample loop of a six-port valve. Four types of wires, including the pristine hydrophobic brass wire, the hydrophilic wire after chemical etching, and both superhydrophobic wires, were comparatively applied to the extraction of six estrogens. The optimized extraction conditions were a sample volume of 60 mL, an injection rate of 2 mL/min, and a desorption time of 2 min at a flow rate of 1 mL/min. The results showed that the highest estrogen extraction efficiency was obtained using the superhydrophobic wire modified by NT, with the enrichment factors in the range of 36–350. Furthermore, the superhydrophobic NT wire exhibited a higher extraction efficiency than the ODT wire with identical superhydrophobicity. This demonstrated that the higher extraction efficiency was mainly dependent on π–π interactions between the sorbent containing naphthalene rings and the target compounds containing benzene rings, rather than surface wettability.

Review of the Real and Sometimes Hidden Costs in Proteomics Experimental Workflows.

A typical proteomics workflow covers all the steps from growing or collecting the cells/tissues/organism, protein extraction, digestion and cleanup, mass spectrometric analysis, and, finally, extensive bioinformatics to derive biological insight from the data. The details of the procedures employed for this can vary widely by laboratory and by sample type: e.g., hard tissues or cells with walls require much more mechanical disruption to extract proteins than do soft tissues, biological fluids, or wall-less cells. Everything then converges on the mass spectrometer, where there are further choices to be made about how to do the analysis. There is one commonality, however, virtually every group around the world now uses liquid chromatography on-line coupled to tandem mass spectrometry, which means that significant amounts of instrument time are dedicated to every sample. There are many other reviews or methods papers, including in this volume, that cover the details of the various procedures involved in proteomic analyses of all types of samples. Our focus here will be on the cost considerations for such analyses, including considerations to ensure that useful data can be obtained the first time a sample is analyzed. Some of these costs are often overlooked, particularly for those groups who operate their own mass spectrometer(s) and do not have to go to a fee-for-service facility to have something analyzed. The chapter presents several challenges and key suggestions in proving hypotheses in proteomics experimental workflow in different biological systems with specific regard to the costs involved, both real and hidden. The detailed methodology for cost-based studies reported in this chapter can help researchers to set up their laboratory with appropriate equipment as well as to identify potential collaborations based on their analytical instrumentation.

Systematic assessment of process analytical technologies for biologics.

The application of process analytical technology (PAT) for biotherapeutic development and manufacturing has been employed owing to technological, economic, and regulatory advantages across the industry. Typically, chromatographic, spectroscopic, and/or mass spectrometric sensors are integrated into upstream and downstream unit operations in in-line, on-line, or at-line fashion to enable real-time monitoring and control of the process. Despite the widespread utility of PAT technologies at various unit operations of the bioprocess, a holistic business value assessment of PAT has not been well addressed in biologics. Thus, in this study, we evaluated PAT technologies based on predefined criteria for their technological attributes such as enablement of better process understanding, control, and high-throughput capabilities; as well as for business attributes such as simplicity of implementation, lead time, and cost reduction. The study involved an industry-wide survey, where input from subject matter industry experts on various PAT tools were collected, assessed, and ranked. The survey results demonstrated on-line liquid Chromatography (LC), in-line Raman, and gas analysis techniques are of high business value especially at the production bioreactor unit operation of upstream processing. In-line variable path-length UV/VIS measurements (VPE), on-line LC, multiangle light scattering (MALS), and automated sampling are of high business value in Protein A purification and polishing steps of the downstream process. We also provide insights, based on our experience in clinical and commercial manufacturing of biologics, into the development and implementation of some of the PAT tools. The results presented in this study are intended to be helpful for the current practitioners of PAT as well as those new to the field to gauge, prioritize and steer their projects for success.

Development of a fast, online three-phase electroextraction hyphenated to fast liquid chromatography–mass spectrometry for analysis of trace-level acid pharmaceuticals in plasma

Sample preparation is a challenge for high-throughput analysis, especially for volume-limited samples with low-abundant analytes. Ideally, sample preparation enriches the analytes of interest while removing the interferents to reduce the matrix effect and improve both sensitivity and quantification. In this study, a three-phase electroextraction (EE) method hyphenated to fast online liquid chromatography–mass spectrometry (LC-MS) was developed. Four model acidic drugs of relevance for drug monitoring in plasma, i.e. naproxen, fenoprofen, flurbiprofen, and ibuprofen, were utilized for the optimization and evaluation of the method. A Design of Experiment approach (Box-Behnken design) was used to optimize the critical parameters of the method, i.e., the type of organic solvent, pH of the sample and acceptor phase, and the extraction voltage and time. Good fitness (P < 0.02, R2 > 0.95) was observed for the developed quadratic model. Extraction could be achieved in less than 2 min (115 s) with enrichment factors (EF) up to 190 and extraction recoveries (ER) up to 38% for academic samples. Additionally, the optimized three-phase EE method was successfully applied to spiked plasma samples with low-abundant (50 ng mL−1) analytes and a low sample volume of 15 μL plasma in 10-fold diluted samples. Finally, two crucial contributors to the matrix effect of three-phase EE application on plasma samples were determined. Specifically, the ion-suppression effect in the MS source was reduced by the fast LC separation, and the matrix effect during extraction was negligible for the diluted protein-precipitated plasma samples. The developed three-phase EE method is easy to operate and provides fast and online extraction of trace-level acidic analytes from volume-limited biological samples. Therefore, this method can provide a potential solution for sample-preparation bottlenecks in high-throughput bioanalysis workflows.