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Five in vitro physicochemical systems have been evaluated in terms of its ability to emulate the skin permeation of neutral compounds: the permeation in two different PAMPA membranes, the classical octanol-water partition coefficient, and two biomimetic chromatography systems, one based in cerasome electrokinetic chromatography and another based in reversed-phase liquid chromatography measurements. The coefficients of the solvation parameter model equation of the mentioned systems have been compared to the ones of the skin permeation process through different comparison parameters. Moreover, a method to predict whether a physicochemical system is able to emulate satisfactorily a biological one, just by the analysis of the equation coefficients has been developed. Results reveal that the two PAMPA systems are a good choice to emulate directly the skin permeation of neutral compounds. Instead, the other three systems need a volume correction term to provide a satisfactory emulation. However, after the correction, all the evaluated systems show a similar ability to emulate well skin permeation, as predicted., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

Developing a knob-into-hole asymmetric bispecific IgG1 monoclonal antibody (mAb) poses manufacturing challenges due to the expression of chain pairing variants, also called mispaired species, in the desired product. The incorrect pairing of light and heavy chains could result in heterogeneous mispaired species of homodimers, heterodimers, light chain swapping, and low molecular weight species (LMWS). Standard chromatography, capillary electrophoretic, or spectroscopic methods poorly resolve these from the main variants. Here, we report a highly sensitive reverse-phase polyphenyl ultra-high-performance liquid chromatography (RP-UHPLC) method to accurately measure mispaired species of Duet mAb format, an asymmetric IgG1 bispecific mAb, for both process development and quality control analytical tests. Coupled with electrospray ionization mass spectrometry (ESI-MS), it enabled direct online characterization of mispaired species. This single direct assay detected diverse mispaired IgG-like species and LMWS. The method resolved eight disulfide bonds dissociated LMWS and three mispaired LMWS. It also resolved three different types of IgG-like mispaired species, including two homodimers and one heterodimer. The characterization and quantification simultaneously enabled the cell line selection that produces a lesser heterogeneity and lower levels of mispaired species with the desired correctly paired product. The biological activity assessment of samples with increased levels of these species quantified by the method exhibited a linear decline in potency with increasing levels of mispaired species in the desired product. We also demonstrated the utility of the technique for testing in-process intermediate materials to determine and assess downstream purification process capability in removing diverse mispaired IgG-like species and LMWS to a certain level during the downstream purification process. Our investigation demonstrates that adopting this method was vital in developing asymmetric bispecific mAb from the initial stage of cell line development to manufacturing process development. Therefore, this tool could be used in the control strategy to monitor and control mispaired species during manufacturing, thus improving the quality control of the final product., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: ‘All authors are or were employees of AstraZeneca and may hold stock ownership or interests in the company. Yasunori Watanabe, Lydia Dewis, and Nikola Lonska were employees of AstraZeneca during experiments and manuscript compilation. reports financial support was provided by AstraZeneca UK Limited. All authors are or were employees of AstraZeneca and may hold stock ownership or interests in the company. Yasunori Watanabe, Lydia Dewis, and Nikola Lonska were employees of AstraZeneca during experiments and manuscript compilation. reports a relationship with AstraZeneca UK Limited that includes: employment. All authors are or were employees of AstraZeneca and may hold stock ownership or interests in the company. Yasunori Watanabe, Lydia Dewis, and Nikola Lonska were employees of AstraZeneca during experiments and manuscript compilation. If there are other authors, they declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.’., (Copyright © 2024 Elsevier B.V. All rights reserved.)

An accurate and efficient method was developed for the determination of azaspiracid shellfish toxins (azaspiracids-1, -2, and -3), neurotoxic shellfish toxins (brevetoxins-2 and -3), diarrhetic shellfish toxins (okadaic acid and dinophysistoxins-1 and -2), and the amnesic shellfish toxin (domoic acid) in mussels (Mytilus galloprovincialis). Lipophilic marine biotoxins (azaspiracids, brevetoxins, and okadaic acid group) were extracted with 0.5 % acetic acid in methanol under heating at 60°C to improve the extraction efficiency of okadaic acid group toxins and then cleaned up with a C18 solid-phase extraction cartridge. Domoic acid was extracted with 50 % aqueous methanol and then cleaned up with a graphitized carbon solid-phase extraction cartridge. Lipophilic marine biotoxins and domoic acid were quantified by reversed-phase liquid chromatography coupled to electrospray ionization tandem mass spectrometry. The developed method had insignificant matrix effects for the nine analytes and good recoveries in the range of 79.0 % to 97.6 % at three spiking levels for all analytes except brevetoxin-2 (43.8-49.8 %). The developed method was further validated by analyzing mussel tissue certified reference materials, and good agreement was observed between certified and determined values., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Semicarbazide, as a derivative of urea, constitutes a great variety of functional molecules for different needs. Herein, novel stationary phases with an incorporated semicarbazide group were proposed. Using aliphatic (docosanoyl, C22) and aromatic (benzoyl, Bz) hydrazides, the semicarbazide-embedded ligands were synthesized before chemical modification of the silica gel, allowing for an accurate interpretation of the chromatographic properties of the corresponding packings. The new stationary phases were water-wettable, due to the presence of highly polar groups. In particular, Bz-semicarbazide (Bz-SCD) stationary phase was sufficiently hydrophilic to run in hydrophilic interaction (HILIC) mode, whilst the C22 (C22-SCD) equivalent, in spite of its reversed-phase nature, was markedly less hydrophobic than the referenced polar-embedded ones. The versatility of C22-SCD was demonstrated with a large selection of analytes, including geometric isomers and standard mixtures of polycyclic aromatic hydrocarbons, sulfonamides, sulfonylurea, substituted ureas, pyridines and carbamates, fat-soluble colorants, antifungal metabolites, angiotensin II receptor blockers and calcium channel blockers., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024. Published by Elsevier B.V.)

Organic UV filters (UVFS) are used to mitigate the dermal effects associated with health risks from UV radiation, making them essential in personal care products. UVFS are frequently identified in environmental samples due to their high lipophilicity and persistence, underscoring the urgency of comprehensive assessments and regulatory measures aimed at safeguarding ecosystems and human health. The present study reports a multiclass analytical method for determining 16 UV sunscreens and metabolites in breast milk based on an ultrasound-assisted-dispersive liquid-liquid micro-extraction (UA-DLLME) with further chromatographic and chemometric resolution. The experimental conditions of the UA-DLLME were optimized through the implementation of the Design of Experiment tools. To model the responses, least-squares and artificial neural network methodologies were implemented. The optimal conditions were found by employing the desirability function. The samples were analyzed through reverse-phase liquid chromatographic separation, UV diode array, and fast-scanning fluorescence detection. The chromatographic analysis enabled the resolution of 16 analytes in a total time of 13.0 min. Multivariate curve resolution-alternating least-square (MCR-ALS) modelling was implemented to resolve analytes that were not fully resolved and to determine analytes that coeluted with endogenous components of the breast milk samples. An enrichment factor of 5-fold concentration was obtained with this methodology, reaching recoveries between 65 % and 105 % for 13 multiclass UV sunscreens and metabolites in breast milk samples with RSD % and REP % lower than 12 %., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier B.V. All rights reserved.)

The current research involved the development and validation of an easy, cost-effective, and sensitive bioanalytical reverse-phase high-performance liquid chromatography method for the assessment of palbociclib (PAL) in rat plasma and kidney, liver, spleen and heart. A response surface methodology-based Box-Behnken design was used to optimize critical chromatographic conditions such as buffer pH, organic phase concentration and flow rate to attain good sensitivity, tailing factor and retention time. The conditions were: pH of buffer, 4.5; organic phase concentration, 40%; Shimpac column with 1.0 ml/min flow rate. The responses were: tailing factor, 1.29 ± 0.03, sensitivity, 366,593 ± 8,592; and retention time, 4.5 ± 0.05 min. The samples were extracted by a protein precipitation method, and absolute recoveries were in the range of 88.99-95.08%. The linearity of the developed method was validated over the range 100-2,000 ng/ml (r 2  ≥ 0.994) in all tested matrices. The developed bioanalytical method showed greater accuracy (0.98 and 4.01%) and precision (<4.88%). The method was optimized for the sensitive analysis of the PAL in rat plasma, and the kidney, liver, spleen and heart were effectively applied to pharmacokinetic studies., (© 2023 John Wiley & Sons Ltd.)

The (bio)pharmaceutical industry is rapidly moving towards complex drug modalities that require a commensurate level of analytical enabling technologies that can be deployed at a fast pace. Unsystematic method development and unnecessary manual intervention remain a major barrier towards a more efficient deployment of meaningful analytical assay across emerging modalities. Digitalization and automation are key to streamline method development and enable rapid assay deployment. This review discusses the use of computer-assisted multifactorial chromatographic method development strategies for fast-paced downstream characterization and purification of biopharmaceuticals. Various chromatographic techniques such as reversed-phase liquid chromatography (RPLC), hydrophilic interaction liquid chromatography (HILIC), ion exchange chromatography (IEX), hydrophobic interaction chromatography (HIC), and supercritical fluid chromatography (SFC) are addressed and critically reviewed. The most significant parameters for retention mechanism modelling, as well as mapping the separation landscape for optimal chromatographic selectivity and resolution are also discussed. Furthermore, several computer-assisted approaches for optimization and development of chromatographic methods of therapeutics, including linear, nonlinear, and multifactorial modelling are outlined. Finally, the potential of the chromatographic modelling and computer-assisted optimization strategies are also illustrated, highlighting substantial productivity improvements, and cost savings while accelerating method development, deployment and transfer processes for therapeutic analysis in industrial settings., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to inappropriately influence the work reported in this paper., (Copyright © 2023 Elsevier B.V. All rights reserved.)

In vitro transcription (IVT) of mRNA is a versatile platform for a broad range of biotechnological applications. Its rapid, scalable, and cost-effective production makes it a compelling choice for the development of mRNA-based cancer therapies and vaccines against infectious diseases. The impurities generated during mRNA production can potentially impact the safety and efficacy of mRNA therapeutics, but their structural complexity has not been investigated in detail yet. This study pioneers a comprehensive profiling of IVT mRNA impurities, integrating current technologies with innovative analytical tools. We have developed highly reproducible, efficient, and stability-indicating ion-pair reversed-phase liquid chromatography and capillary gel electrophoresis methods to determine the purity of mRNA from different suppliers. Furthermore, we introduced the applicability of microcapillary electrophoresis for high-throughput (<1.5 min analysis time per sample) mRNA impurity profiling. Our findings revealed that impurities are mainly attributed to mRNA variants with different poly(A) tail lengths due to aborted additions or partial hydrolysis and the presence of double-stranded mRNA (dsRNA) byproducts, particularly the dsRNA 3'-loop back form. We also implemented mass photometry and native mass spectrometry for the characterization of mRNA and its related product impurities. Mass photometry enabled the determination of the number of nucleotides of different mRNAs with high accuracy as well as the detection of their size variants [i.e., aggregates and partial and/or total absence of the poly(A) tail], thus providing valuable information on mRNA identity and integrity. In addition, native mass spectrometry provided insights into mRNA intact mass, heterogeneity, and important sequence features such as poly(A) tail length and distribution. This study highlights the existing bottlenecks and opportunities for improvement in the analytical characterization of IVT mRNA, thus contributing to the refinement and streamlining of mRNA production, paving the way for continued advancements in biotechnological applications.

The present study focuses on the development of a simple, rapid, specific, and stability-indicating HPLC method for the simultaneous analysis of pyridostigmine bromide (PGB) and sodium benzoate (SBN) in oral liquid dosage forms. Analytical techniques should enhance sensitivity and specificity for the estimation of pharmaceutical drug products. Stress studies were conducted under various International Conference on Harmonization (ICH) conditions for evaluation. The further optimized HPLC method was validated in accordance with the current ICH guidelines. Chromatographic separation was accomplished using a mobile phase consisting of a 950:50 v/v ratio of perchloric acid buffer and acetonitrile as mobile phase-A, and 100% acetonitrile as mobile phase-B. The flow rate is 1.0 mL/min, and the injection volume is 20 μL. Detection of components was carried out at 220 nm for PGB and 228 nm for SBN. The validated HPLC method demonstrated high specificity, with linearity ranging between 24 and 72 μg/mL for PGB and 5.2-15.6 μg/mL for SBN. The correlation coefficient for both drugs exceeded 0.999. The method demonstrated high accuracy, exceeding 97%. In stress studies, PGB was found to be sensitive to alkaline stress conditions. The results reveal the successful applicability of the current method for the estimation of PGB and SBN in its marketed formulation, which can be reasonably inferred to assess other formulation systems., (© 2023 John Wiley & Sons, Ltd.)

A reversed-phase high-performance liquid chromatographic (HPLC) method was developed for the simultaneous determination of the potential impurities of dexketoprofen, including the distomer R-ketoprofen. After screening the separation capability of four polysaccharide columns (Lux Amylose-1, Lux Amylose-2, Lux Cellulose-1 and Lux Cellulose-2) in polar organic and in reversed-phase modes, appropriate enantioseparation was observed only on the Lux Amylose-2 column in an acidified acetonitrile/water mixture. A detailed investigation of the mobile phase composition and temperature for enantio- and chemoselectivity showed many unexpected observations. It was observed that both the resolution and the enantiomer elution order can be fine-tuned by varying the temperature and mobile phase composition. Moreover, hysteresis of the retention times and enantioselectivity was also observed in reversed-phase mode using methanol/water mixtures on amylose-type columns. This could indicate that the three-dimensional structure of the amylose column can change by transitioning from a polar organic to a reversed-phase mode, which affects the enantioseparation process. Temperature-dependent enantiomer elution order and rare enthalpic/entropic controlled enantioseparation in the operative temperature range were also observed in reversed-phase mode. To find the best methodological conditions for the determination of dexketoprofen impurities, a full factorial optimization design was performed. Using the optimized parameters (Lux Amylose-2 column with water/acetonitrile/acetic acid 50/50/0.1 ( v / v / v ) at a 1 mL/min flow rate at 20 °C), baseline separations were achieved between all compounds within 15 min. Our newly developed HPLC method was validated according to the current guidelines, and its application was tested on commercially available pharmaceutical formulations. According to the authors' knowledge, this is the first study to report hysteretic behavior on polysaccharide columns in reversed-phase mode.

A method was developed for the determination of 26 drugs of abuse from different classes, including illicit drugs in quantitative dried blood spots (qDBSs), with the aim to provide a convenient method for drug testing by using only 10 μL of capillary blood. A satisfactory limit of quantification (LOQ) of 2.5 ng/mL for 9 of the compounds and 5 ng/mL for 17 of the compounds and a limit of detection (LOD) of 0.75 ng/mL for 9 of the compounds and 1.5 ng/mL for 17 of the compounds were achieved for all analytes. Reversed-phase liquid chromatography was applied on a C18 column coupled to MS, providing selective detections with both +ESI and -ESI modes. Extraction from the qDBS was performed using AcN-MeOH, 1:1 ( v / v ), with recovery ranging from 84.6% to 106%, while no significant effect of the hematocrit was observed. The studied drugs of abuse were found to be stable over five days under three different storage conditions (at ambient temperature 21 °C, at -20 °C, and at 35 °C), thus offering a highly attractive approach for drug screening by minimally invasive sampling for individuals that could find application in forensic toxicology analysis.

In the present work, comprehensive two-dimensional reversed-phase countercurrent chromatography × reversed-phase liquid chromatography combined (2D RPCCC × RPLC) with 2D microfraction bioactive evaluation was employed to screen and isolate α-glucosidase inhibitors from Rheum palmatum L. Countercurrent chromatography was employed to improve 2D analysis and preparative separation. A selected biphasic solvent system composed of petroleum ether/ethyl acetate/methanol/water with gradient elution mode was used for the first dimension RPCCC separation ( 1 D RPCCC). Solid-phase extraction was applied to eliminate interfering polar compounds before the second dimension analysis ( 2 D RPLC). 76 components were shown in 2D contour plot in UV 280 nm. 11 Candidates were separated by a scaled-up CCC and identified by 1 H NMR and 13 C NMR, including anthraquinones, flavonoids, stilbenes, phenols, and glucoside derivatives. In addition, it was found that two components, resveratrol-4'-O-(6″-galloyl)glucoside (36) and lyciumaside (43) were identified as natural α-glucosidase inhibitors in Rheum palmatum L. for the first time., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Hydroxy- and peroxy-triacylglycerols are common products of lipid peroxidation formed during oil storage or heating or as enzymatic oxidation product of arachidonic acid as signaling molecules in mammals. In this study, oxygenated triacylglycerides (TAG) were identified in pistachio oil based on reverse phase(RP), high-performance liquid chromatography coupled with electrospray ionization and mass spectrometry (HPLC- ESI -MS). 20 novel lipid plant metabolites, classified based on their fragment spectra into a hydroxy (TAG-OH), an epoxy (TAG-O), and hydroperoxide (TAG-OOH) groups. We believe that this class of compounds has been for the first time observed as genuine secondary plant metabolites in a natural source in this case pistachio lipids of dietary relevance., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

Background: Therapeutic monoclonal antibodies (mAbs) comprise a large structural variability with respect to charge, size and post-translational modifications. These critical quality attributes (CQAs) need to be assessed during and after the production of mAbs. This normally requires off-line purification and sample preparation as well as several chromatographic selectivities, which makes the whole process time-consuming and error-prone. To improve on this, we developed an integrated and automated multi-dimensional analytical platform for the simultaneous assessment of multiple CQAs of mAbs in cell culture fluid (CCF) from upstream processes., Results: The on-line system allows mAb characterization at the intact level, combining protein A affinity chromatography (ProtA) with size-exclusion, ion-exchange, and reversed-phase liquid chromatographic modes with UV and mass spectrometric detection. Multiple heart cuts of a single mAb elution band from ProtA are stored in 20-μL loops and successively sent to the multimethod options in the second dimension. ProtA loading and elution conditions and their compatibility with second-dimension LC modes were studied and optimized. Subsequently, heart-cutting and valve-switching schemes were investigated to achieve effective and reproducible analyses. The applicability of the developed workflow was demonstrated by the direct analysis (i.e. not requiring off-line sample preparation) of a therapeutic mAb in CCF, obtaining useful information on accurate molecular mass, glycosylation, and charge and size variants of the mAb product at the same time and in just over 1 h., Significance: The developed multidimensional platform is the first system that allows for multiple fractions from a single ProtA band to be characterized using different chromatographic selectivities in a single run allowing direct correlation between CQAs. The performance of the system is comparable to established off-line methods, fully compatible with upstream process samples, and provides a significant time-reduction of the characterization procedure., Competing Interests: Declaration of competing interest The authors declare the following financial interests/ which may be considered as potential competing interests: Govert W. Somsen reports equipment was provided by Agilent Technologies Inc., (Copyright © 2023 The Authors. Published by Elsevier B.V. All rights reserved.)

Bioactive peptide-based drugs have gained exceeding attention as promising treatments for infectious and oxidative-stress-related diseases, are exacerbated by the advent and spread of various multidrug-resistant bacteria and industrial lifestyles. Fish skin mucus has been recognized as a potential source of bioactive peptides, providing the first line of fish defense against invading pathogens which are targeted here to be explored as a new source of biopharmaceutics. Peptide fractions were isolated from the epidermal exudates of Caspian sand goby, Neogobius fluviatilis pallasi, by solid-phase extraction (SPE), ultrafiltration, and reversed-phase chromatography. The resulting fractions were characterized for their antibacterial and antioxidant properties, and results showed that the molecular weight fraction < 5 kDa represented the highest (p < 0.05) bacterial inhibition activity against Staphylococcus aureus and Bacillus subtilis as well as scavenging activity against DPPH and ABTS radicals. Overall, these results introduce the epidermal mucus of Caspian sand goby as a valuable source of bioactive compounds that can be considered new and efficient biopharmaceutics., (© 2024. The Author(s).)

The development of quantitative structure-retention relationship (QSRR) models has, until recently, required an adequate selection of molecular descriptors necessarily obtained based on a known chemical structure. However, these complex descriptors are not always available nor calculable when the high-resolution mass spectrometry (HRMS) annotation process is underway. Depending on the level of annotation, many structures or even various molecular formulas could be candidates. To secure and improve the annotation process and to save time, a QSRR model (using only 0D molecular descriptors) to predict retention times in reverse-phase liquid chromatography (RPLC) based on the molecular formula was developed, and a general QSRR annotation-based methodology was also proposed., Competing Interests: Declaration of competing interest The authors declare that they have no conflicts of interest., (Copyright © 2023 Elsevier B.V. All rights reserved.)

Zwitterions are a promising choice to prepare separation materials because of their hydrophilicity and biocompatibility. We described the preparation of two zwitterionic polymer functionalized stationary phases and evaluation under mixed-mode chromatography. A zwitterionic monomer, S-(4-vinylbenzyl) cysteine (SVC), was synthesized and bonded to silica via reversible addition fragmentation chain transfer (RAFT) polymerization to afford a zwitterionic stationary phase, Sil-SVC. A hydrophobic monomer, N-(4-phenylbutan-2-yl) acrylamide (NPA), was copolymerized with SVC onto the stationary phase (Sil-SVCNPA) for comparison. The stationary phases were characterized with FT-IR, TGA, EA, and zeta-potential measurements. Mobile phase composition (ACN content, pH and salt concentration) was varied to study the retention property. Linear solvation energy relationship and Van't Hoff plot were used to investigate the retention mechanism and how chromatographic conditions influenced it. Both stationary phases showed a mixed-mode of RPLC/HILIC/IEC and satisfactory performance in separating hydrophobic analytes (alkylbenzenes and polycyclic aromatic hydrocarbons), hydrophilic nucleotide and bases, and anions, high column efficiency of 60,000 plates·m -1 was achieved. In summary, zwitterionic polymers are attractive options to prepare stationary phases and the retention property can be easily regulated by copolymer., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier B.V. All rights reserved.)

Vemurafenib (VMF) is a practically insoluble (< 0.1 μg/mL) and least bioavailable (1%) drug. To enhance its oral bioavailability and solubility, we formulated a reliable self-nano emulsifying drug delivery system (SNEDDS). A Quality by Design (QbD) approach was used to optimize the ratio of Capryol 90, Tween 80, and Transcutol HP. VMF-loaded SNEDDS was characterized for its size, polydispersity index (PDI), zeta potential, drug content, and transmittance. The in vitro release profile of the drug loaded in SNEDDS was compared to the free drug in two media, pH 6.8 and 1.2, and the data obtained were analyzed with different mathematical models. A reverse-phase ultra-pressure liquid chromatography (UPLC) technique with high sensitivity and selectivity was developed and validated for the quantification of VMF in analytical and bioanalytical samples. Dissolution efficiency for SNEDDS was estimated using different models, which proved that the developed novel SNEDDS formulation had a better in vitro dissolution profile than the free drug. A 2.13-fold enhanced oral bioavailability of VMF-loaded SNEDDS compared to the free drug demonstrates the superiority of the developed formulation. This work thus presents an overview of VMF-loaded SNEDDS as a promising alternative to improve the oral bioavailability of the drug., (© 2024. The Author(s), under exclusive licence to American Association of Pharmaceutical Scientists.)

Lipids are key constituents of the barrier function in the human stratum corneum (SC), which is the outermost layer of the epidermis and amenable to non-invasive sampling by tape stripping. The three major lipid classes in the SC, i.e., ceramides, fatty acids, and cholesterol, present equimolar concentration. Liquid chromatography coupled with mass spectrometry (LCMS) is elective in profiling lipids in the SC in both positive and negative ion modes. Nevertheless, the latter one allows for the simultaneous detection of the three major epidermal components of the SC. Determination of ceramides in the SC poses analytical challenges due to their wide range of structures and concentrations especially in the case of limited sample amounts. Ammonium formate is a commonly used modifier added to the mobile phase to assist ionization. However, it introduces uncertainty in the identification of ceramides when operating in negative ion mode, even with high resolution MS. We tested the advantages of using fluoride in the lipid profiling of SC and unambiguous identification of ceramides subclasses. The use of fluoride enhanced the ionization of ceramides, regardless the specific substructure, solved misidentification issues, and was successfully applied to the simultaneous detection of all three lipid classes in the human SC., (© 2024. The Author(s).)

Oxylipins - involved in inflammatory processes - are reported in several diseases, in biological, pharmacological, and physiological fields. To face the structural complexity of oxylipins, the study of isomers and isobars species relied on Selected Reaction Monitoring (SRM) and Multiple Reaction Monitoring (MRM) in tandem mass spectrometry such as triple quadrupole, quadrupole-Time of Flight (TOF). Unfortunately, false positive signals in cellular matrix could occur using MRM or SRM mode since the MS/MS spectrum of each molecule is not acquired with the previous mode to help molecule confirmation. Using the versatile ability of LTQ-Orbitrap® Velos Pro mass spectrometer, we developed a novel method based on data dependent acquisition (DDA) workflow for oxylipins analysis. To reach sufficient data points per peak and a better sensitivity to quantify oxylipins traces, an optimization of the acquisition frequency was carried out both on linear trap and Orbitrap analyzers. A segmentation of the chromatographic profile and an optimization of the collision energies by HCD (higher energy collision dissociation) for each eicosanoid increased the acquisition frequency significantly and the detection threshold: around 2 pg for some prostanoids and 0.02-2 pg for some leukotrienes and oxidized species. We validated our method in terms of specificity (RSD <10%), sensitivity, accuracy and precision. The intra and inter-day accuracy were between 86.56% and 114.93%. Besides, a relative standard deviation less than 15% as intra- and inter-day precision were obtained for almost all molecules. A linear range between 2.5 and 12,500 pg was reached. DDA approach on LTQ-Orbitrap® constitutes an alternative to MRM mode on triple quadrupole for eicosanoids quantification in complex matrices. Finally, this method helped us to compare for the first time the amount of prostanoids released by J774 and THP-1 macrophages under lipopolysaccharide (LPS) stimulation., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier B.V. All rights reserved.)

Ion concentration in liposomal drugs is important for drug stability and drug release profile. However, quantifying ion concentration in liposomal drugs is challenging due to the absence of chromophores or fluorophores of ions and the efficiency of their release from the liposome structure. To address these issues, a method based on reversed-phase high-performance liquid chromatography (RP-HPLC) coupled with a charged aerosol detector (CAD) has been developed to determine total, internal, and external ions in drug-loaded liposomal products. In this protocol, we focused on the quantitation of ammonium and sulfate ions in liposomal products, using generic PEGylated liposomal doxorubicin as an example. This method can be extended to calcium, acetate, and other ions in different liposomal formulations with slight modifications., (© 2024. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

In recent years a multitude of LC-MS/MS assays have been widely reported in commercial and clinical literature demonstrating the simultaneous analyses of dozens of drugs of abuse in human samples. The utility of such assays is meant to supplant the indirect detection based on the classical spectral library approach. Direct and simultaneous analysis via LC-MS/MS technology is made possible by fast acquisition rates in multiple reaction monitoring, as well as sensitivity and high selectivity of the technology for each individual analyte in a complex mixture. Hence, unlike immunoassays, which are not well-suited for the analyses of mixtures, and which may also be prone to false positives from potential interferences, quantitative LC-MS/MS analyses are feasible for complex patient mixtures of drugs of abuse. We hereby present a robust clinical LC-MS/MS assay for the simultaneous and semi-quantitative analysis of up to 62 drugs of abuse in human urine, representing major classes that include opiates, benzodiazepines, amphetamines, etc. The assay utilizes dilute and shoot, whereby the sample is diluted ten times in internal standard reagent and thereafter submitted to the LC-MS instrument, i.e., reversed-phase liquid chromatography coupled to the electrospray ionization multiple reaction monitoring analysis, via the TSQ Endura triple-quadrupole instrument. The assay employs stable isotope-labeled internal standards with a linear response in the 30-300 ng/mL range, effectively semi-quantitative, since this analytical range is well within typical immunoassay cutoffs for most drugs., (© 2024. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

The comprehensive and efficient characterization of components in traditional Chinese medicine is crucial for elucidating its active constituents and uncovering its mechanism. Identifying the compounds of the Bushen Huoxue Prescription (BHP) is difficult because of its complex composition and the large difference in concentration among its compounds. In this study, a hydrophilic interaction liquid chromatography coupled with reversed-phase LC (HILIC × RPLC) offline 2D-LC tandem high-resolution mass spectrometry method was established to analyze the total compounds of the BHP. Database screening and molecular networking were performed to identify the compounds. In contrast to conventional 1D chromatography, 2D chromatography increased peak capacity, enriched trace ingredients, and prevented the masking of high-abundance compounds. A total of 165 compounds were identified, and 14 potential compounds needed to be further identified. This study provided an effective method for comprehensively analyzing the complex system of traditional Chinese medicine compounds., (© 2024 Wiley-VCH GmbH.)

We hereby present a fast, high-throughput, and clinical LC-MS/MS assay for the simultaneous analysis of benzodiazepines in human urine. The assay is used as both a confirmations and semi-quantitative assay for the abovementioned drugs of abuse following immunoassay urine drug screens. Urine levels are reported in the range of 25 ng/mL-500 ng/mL for each of the 22 analytes, based on a six-level calibration and using a subset (10) of stable isotopically labeled analogues as internal standards. The urine sample is clarified, diluted ten times in internal standard reagent, and thereafter injected into the LC-MS/MS instrument. Reversed-phase liquid chromatography is used to separate the mixture, and the TSQ Endura triple-quadrupole (QqQ) MS instrument performs detection via positive-mode electrospray ionization multiple reaction monitoring., (© 2024. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

The integration of complementary analytical platforms is nowadays the most common strategy for comprehensive metabolomics analysis of complex biological systems. In this chapter, we describe methods and tips for the application of a mass spectrometry multi-platform in Alzheimer's disease research, based on the combination of direct mass spectrometry and orthogonal hyphenated approaches, namely, reversed-phase ultrahigh-performance liquid chromatography and gas chromatography. These procedures have been optimized for the analysis of multiple biological samples from human patients and transgenic animal models, including blood serum, various brain regions (e.g., hippocampus, cortex, cerebellum, striatum, olfactory bulbs), and other peripheral organs (e.g., liver, kidney, spleen, thymus)., (© 2024. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Analytical scale purification of rare earth element (REE) radioisotopes is typically accomplished using cation-exchange resins (e.g. AG 50W-X8) and high-performance liquid chromatography (HPLC). Despite the variety of improvements made since the development of this separation process in the 1950s, nearest neighbor separations remain a challenge, as does the issue of irreversible sample adsorption. Herein, we report a study that evaluates the potential of high-speed counter-current chromatography (HSCCC) as an alternative method for purifying REE elements, with specific reference to separations of fission product REE of interest to nuclear forensics. Complementary HSCCC REE separation experiments, one spiked with radiotracer and REE fission product activity, allowed for in depth analysis of resulting fractions from both an elemental (inductively coupled plasma atomic emission spectroscopy, ICP-AES) and radiological (gamma-ray spectrometry, beta counting) purity perspective. The highly reproducible nature of separation profiles generated from HSCCC instruments was leveraged to simplify work-up of samples containing radioisotopes. Subsequent radioanalytical evaluation revealed minimal carryover of Eu into neighboring Sm and Tb fractions (as indicated by presence of 150 Eu), and trace contamination of the Tb fraction with Y (as indicated by presence of 91 Y). Subtle differences in stationary phase retention across the two columns were reflected in significant variations in decontamination factors of duplicate parallel separations. These differences paired with obtained distribution of radioisotopes provided valuable insights into future improvements. Collectively, this study represents a significant step forward in development of HSCCC technology for task specific REE radioisotope purification., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023. Published by Elsevier B.V.)

Alzheimer's disease (AD) is the most prevalent form of dementia, disproportionately affecting women in disease prevalence and progression. Comprehensive analysis of the serum proteome in a common AD mouse model offers potential in identifying possible AD pathology- and gender-associated biomarkers. Here, we introduce a multiplexed, nondepleted mouse serum proteome profiling via tandem mass-tag (TMTpro) labeling. The labeled sample was separated into 475 fractions using basic reversed-phase liquid chromatography (RPLC), which were categorized into low-, medium-, and high-concentration fractions for concatenation. This concentration-dependent concatenation strategy resulted in 128 fractions for acidic RPLC-tandem mass spectrometry (MS/MS) analysis, collecting ∼5 million MS/MS scans and identifying 3972 unique proteins (3413 genes) that cover a dynamic range spanning at least 6 orders of magnitude. The differential expression analysis between wild type and the commonly used AD model (5xFAD) mice exhibited minimal significant protein alterations. However, we detected 60 statistically significant (FDR < 0.05), sex-specific proteins, including complement components, serpins, carboxylesterases, major urinary proteins, cysteine-rich secretory protein 1, pregnancy-associated murine protein 1, prolactin, amyloid P component, epidermal growth factor receptor, fibrinogen-like protein 1, and hepcidin. The results suggest that our platform possesses the sensitivity and reproducibility required to detect sex-specific differentially expressed proteins in mouse serum samples.

Gangliosides are complex lipids found in human milk that play important structural and biological functions. In this study, we utilized reversed-phase liquid chromatography coupled to quadrupole time-of-flight mass spectrometry to evaluate the molecular distribution of GM3 in human milk samples collected at distinct lactation stages, ranging from colostrum to advanced lactation samples. Throughout lactation, GM3 d40:1 emerged as the most abundant GM3 species, except in colostrum, where GM3 d42:2 prevailed. The relative content of GM3 species containing very long N-fatty acyl (N-FA) substituents with >22 carbon atoms decreased, while the content of GM3 species containing 14:0, 18:0, 18:1, and 20:0 N-FA substituents increased in the later months of lactation. These findings highlight the divergence of GM3 profiles across the lactation period. Moreover, considerable interindividual variance was observed among the analyzed samples. The assessment of the GM3 profiles contributes to our understanding of the dynamic composition of human milk.

The present work describes a re-parameterization of the Neue Kuss (NK) model for describing retention in liquid chromatography, and this re-parameterized model is used to fit a large set of isocratic retention measurements with improved convergence properties relative to the original parameterization of the model. Next, an experimental design for retention measurements using mobile phase gradient elution conditions is proposed for the purpose of obtaining accurate and precise NK parameters. Simulated retention data for mobile phase gradient elution conditions with two different levels of noise, as well as an essentially zero noise level were fit with the re-parameterized model. The results showed that the re-parameterized fits yielded average (absolute value) prediction errors for the parameters at the highest noise level of 7.2 % for S 1,ref , 18 % for S 2,ref and 6.2 % for k ref (the re-parameterized NK model parameters). These errors were significantly smaller than those for the original parameterization of the NK model, where the errors were 23 % for S 1 , 25 % for S 2 and 160 % for k w (the original NK model parameters). Furthermore, isocratic retention factors predicted using these model parameters were found to have an average magnitude of error of 0.51 % for the re-parameterized model, as opposed to 6800 % for the model with the original parameterization. A further test of this approach was carried out for independent experimental measurements for five solutes on a C18 column. The average magnitude of error of the isocratic retention factors predicted from parameters obtained from fits of gradient data was 1.6 %, provided that the range of organic solvent compositions that the solute sampled in the mobile phase gradient experiments was consistent with the isocratic experiments. These results indicate that the re-parameterization of the NK model allows for significant improvements in the fitting process, and that the proposed experimental design allows for NK parameters to be extracted from mobile phase gradient experiments, with prediction accuracies of isocratic retention factors on the order of 1-2 %., Competing Interests: Declaration of Competing Interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Dwight R. Stoll reports financial support was provided by National Science Foundation. Dwight R. Stoll reports equipment, drugs, or supplies was provided by Agilent Technologies Inc., (Copyright © 2023 Elsevier B.V. All rights reserved.)

Infigratinib, a protein kinase inhibitor employed in the therapeutic management of cholangiocarcinoma, was subjected to various stress conditions, including hydrolytic (acidic and alkaline), oxidative, photolytic, and thermal stress, in accordance with the rules established by the International Council for Harmonization. A cumulative count of five degradation products was observed. The application of the Quality by Design principle was utilized in the development of a rapid and specific separation method for Infigratinib and its degradation products. The methodology employed in this study was derived from an experimental design approach, which was utilized to examine the critical process parameters associated with chromatographic systems. The reversed-phase high-performance liquid chromatography technique, employing a C18 column and a mobile phase composed of a gradient mixture of 25 mM ammonium acetate buffer at pH 6.0 and acetonitrile, successfully facilitated the chromatographic separation. The methodology was expanded to include the utilization of UPLC-quadrupole tandem mass spectrometry in order to conduct a comprehensive analysis of the structural properties and characterize the degradation products. Overall, five degradation products were found in different stress conditions. The method was verified at certain working points, wherein a linearity range (5.0-200.0 µg/mL) was developed and other parameters such as accuracy, repeatability, selectivity, and system suitability were evaluated. Finally, the toxicity and mutagenicity of Infigratinib and its degradation products were predicted using in silico software, namely DEREK Nexus ® (version 6.2.1) and SARAH Nexus ® (version 3.2.1). Various toxicity endpoints, including chromosomal damage, were predicted. Additionally, two degradation products were also predicted to be mutagenic.

Background: The kynurenine pathway (KP) generates eight tryptophan (TRP) metabolites collectively called kynurenines, which have gained enormous interest in clinical research. The importance of KP for different disease states calls for developing a low-cost and high-throughput chromatography-mass spectrometry method to evaluate the potential of different kynurenines. Simultaneous separation of TRP and its eight metabolites is challenging because they have substantial polarity differences (log P = -2.5 to +1.3)., Results: A low-cost, reversed-phase LC-MS/MS method based on polarity partitioning was established to simultaneously separate and quantitate all nine kynurenine pathway metabolites (KPMs) in a single run for the first time in the open literature. Based on stationary phase screening and ternary mobile phase optimization strategy, high polarity KPMs were retained while medium and low polarity KPMs were eluted in a shorter time. After method validation, we demonstrated the applicability of this LC/MS/MS method by quantitative measurement of all nine KPM in cerebrospinal fluid (CSF) and plasma among two groups of human subjects diagnosed with depression. Furthermore, we measured the differential KPMs in these two groups of low and high inflammation and correlated the results with CRP or TNF-α markers for depression., Significance: Our proposed LC-MS/MS provides a new metabolite assay that can be easily applied in various clinical applications to simultaneously quantify multiple biomarkers in KP dysfunction., Competing Interests: Declaration of competing interest All the authors have declared that there was no competing conflict of interests that may have influenced the research data reported in this paper., (Copyright © 2023 Elsevier B.V. All rights reserved.)

We evaluated the suitability of supercritical fluid chromatography (SFC) for oligonucleotide analysis using 4-mer oligonucleotides with various phosphorothioate (PS) contents as model compounds. Column screening showed that the diol-modified column was able to separate sequences with different PS contents. Optimization of the column body and additives allowed us to analyze polar oligonucleotides using SFC. Various sequences were also analyzed using the optimized method. A good peak shape was obtained when the guanine plus cytosine content of the analyte was two or less in the 4-mer oligonucleotides. Furthermore, we found that the retention times of the selected sequences were positively correlated with polar surface areas, indicating that oligonucleotides interact with polar stationary phases. In contrast, more hydrophobic full PS sequences were retained more strongly in the diol column than the full phosphodiester (PO) sequences. This suggests that the diol column has unique selectivity for PO and PS linkages. These results indicate that SFC is potentially applicable to oligonucleotide analysis with a separation mechanism that is different from that of ion-pair reversed-phase liquid chromatography., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023. Published by Elsevier B.V.)

Reversed-phase liquid chromatography (RPLC) is the analytical tool of choice for monitoring process-related organic impurities and degradants in pharmaceutical materials. Its popularity is due to its general ease-of-use, high performance, and reproducibility in most cases, all of which have improved as the technique has matured over the past few decades. Nevertheless, in our work we still occasionally observe situations where RPLC methods are not as robust as we would like them to be in practice due to variations in stationary phase chemistry between manufactured batches (i.e., lot-to-lot variability), and changes in stationary phase chemistry over time. Over the last three decades several models of RPLC selectivity have been developed and used to quantify and rationalize the effects of numerous parameters (e.g., effect of bonded phase density) on separation selectivity. The Hydrophobic Subtraction Model (HSM) of RPLC selectivity has been used extensively for these purposes; currently the publicly available database of column parameters contains data for 750 columns. In this work we explored the possibility that the HSM could be used to better understand the chemical basis of observed differences in stationary phase selectivity when they occur - for example, lot-to-lot variations or changes in selectivity during column use. We focused our attention on differences and changes in the observed selectivity for a pair of cis-trans isomers of a pharmaceutical intermediate. Although this is admittedly a challenging case, we find that the observed changes in selectivity are not strongly correlated with HSM column parameters, suggesting that there is a gap in the information provided by the HSM with respect to cis-trans isomer selectivity specifically. Further work with additional probe molecules showed that larger changes in cis-trans isomer selectivity were observed for pairs of molecules with greater molecular complexity, compared to the selectivity changes observed for simpler molecules. These results do not provide definitive answers to questions about the chemical basis of changes in stationary phase chemistry that lead to observed differences in cis-trans isomer selectivity. However, the results do provide important insights about the critical importance of molecular complexity when choosing probe compounds and indicate opportunities to develop improved selectivity models with increased sensitivity for cis-trans isomer selectivity., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier B.V. All rights reserved.)

Although various metabolomic methods have been reported in recent years, simultaneous detection of hydrophilic and hydrophobic metabolites in a single analysis remains a technical challenge. In this study, based on the combination of hydrophilic interaction liquid chromatography (HILIC) and reversed phase liquid chromatography (RPLC), an online two-dimensional liquid chromatography/triple quadrupole mass spectrometry method (2D-LC/TQMS) was developed for the simultaneous analysis of hydrophilic and hydrophobic metabolites of various biological samples. The method can measure 417 biologically important metabolites (e.g., amino acids and peptides, pyrimidines, purines, monosaccharides, fatty acids and conjugates, organic dicarboxylic acids, and others) with logP values ranging from -10.3 to 21.9. The metabolites are involved in a variety of metabolic pathways (e.g., purine metabolism, pyrimidine metabolism, tyrosine metabolism, galactose metabolism, gluconeogenesis, and TCA cycle). The developed method has good intra- and inter-day reproducibility (RSD of retention time <2%, RSD of peak area <30%), good linearity (R 2  > 0.9) and wide linear range (from 0.0025 μg/mL to 5 μg/mL). The applicability of the method was tested using different biological samples (i.e., plasma, serum, urine, fecal, seminal plasma and liver) and it was found that 208 (out of 417) identical metabolites were detected in all biological samples. Furthermore, the metabolomic method was applied to a case/control study of urinary of bladder cancer. Thirty differential metabolites were identified that were involved in carbohydrate and amino acid metabolism., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier B.V. All rights reserved.)

Protein methylation is receiving more and more attention due to its essential role in diverse biological processes. Large-scale analysis of protein methylation requires the efficient identification of methylated peptides at the proteome level; unfortunately, a significant number of methylated peptides are highly hydrophilic and hardly retained during reversed-phase chromatography, making it difficult to be identified by conventional approaches. Herein, we report the development of a novel strategy by combining hydrophobic derivatization and high pH strong cation exchange enrichment, which significantly expands the identification coverage of the methylproteome. Noteworthily, the total number of identified methylated short peptides was improved by more than 2-fold. By this strategy, we identified 492 methylation sites from NCI-H460 cells compared to only 356 sites identified in native forms. The identification of methylation sites before and after derivatization was highly complementary. Approximately 2-fold the methylation sites were obtained by combining the results identified in both approaches (native and derivatized) as compared with the only analysis in native forms. Therefore, this novel chemical derivatization strategy is a promising approach for the comprehensive identification of protein methylation by improving the identification of methylated short peptides.

A novel composite hydrogel functionalized silica core-shell stationary phase was prepared by the surface modification of silica sphere. The successful synthesis of the new stationary phase (T-Sil@PAM/SA/UiO-66-NH 2 ) was proven by scanning electron microscope (SEM), transmission electron microscope (TEM), X-ray photoelectron spectroscopy (XPS) and X-ray diffraction (XRD), etc. Due to the coexistence of amide, hydroxyl, long carbon chain and UiO-66-NH 2 in composite hydrogel shell, the obtained stationary phase can be used in hydrophilic/reversed-phase liquid chromatography with multiple retention mechanisms, such as hydrophilic, hydrophobic and π - π interactions. The chromatographic retention behavior of T-Sil@PAM/SA/UiO-66-NH 2 demonstrated that the new stationary phase showed excellent separation performance for both polar analytes (such as alkaloids, saccharides, etc.) and nonpolar analytes (such as substituted benzene and polycyclic aromatic hydrocarbon (PAHs), etc.). Furthermore, compared with NH 2 column and commercial C18 column, the T-Sil@PAM/SA/UiO-66-NH 2 exhibited a certain superiority. Moreover, the relative standard deviation (RSD) of PAHs' retention time with eight replicates consecutive elution was found to range from 0.03% to 0.17%. Therefore, the successful use of T-Sil@PAM/SA/UiO-66-NH 2 in mixed‑mode liquid chromatography expanded the potential applications of hydrogels in the field of separation., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier B.V. All rights reserved.)

For confirmation and/or screening purposes, rapid, selective, and precise chromatographic methods are required. In this vein, the utility of SiH columns (C18, UDC Cholesterol, and Diamond Hydride) with photodiode array UV absorption and single quadrupole MS detection for multi-modal separation of representative drugs from different drug classes on a single column using the same solvent reservoirs was investigated. For a conventional two column approach employing a combination of conventional C18 and silica columns operating in both the reversed phase chromatographic and hydrophilic interaction chromatographic modes, gradient analysis is required for the first column and there is a lack of retention on the second column for non-amine analytes. In comparison, all analytes are retained for two relatively rapid (< 10 min), precise (% RSD <0.4%), and non-correlated isocratic separations (R 2 =0.2115) when using a UDC Cholesterol column., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier B.V. All rights reserved.)