The crucial role of steroid hormones in health and diseases merits their high-throughput, accurate and affordable measurements in biological specimens. Despite advances in analytical methods, sensing ...

Amino acids (AAs) are considered as the building blocks of life. Unlike nonessential AAs, the human body cannot synthesize essential AAs and should be supplied in food or dietary supplements. The aim of the work is simultaneous HPLC-UV determination of 10 structurally related AAs without pre- or postderivatization in powdered dietary supplements (PDSs). This was challenging, especially because PDS has no standardized procedures for its quality control. HPLC-UV chromatograms of the 10 AAs were recorded using a gradient elution of the mobile phase on a CLC-C18 column at 225 nm. The elution started with 100% of phosphate buffer (pH 7.4, 10 mM) for 10 min; then, the concentration of acetonitrile increased linearly to reach 50% for another 15 min at room temperature. Good separation was achieved within a 25 min run time without pre- or postderivatization. The method was carefully validated according to the ICH guidelines over the linearity range of 100–200, 50–200, 20–150, 50–400, 20–250, 75–175, 50–250, 50–250, 50–300, and 5–100 μg/mL for l-lysine, l-threonine, l-histidine, l-valine, l-methionine, l-isoleucine, l-leucine, l-tyrosine, l-phenylalanine, and l-tryptophan, respectively, with mean recoveries ranges between 98.91 and 100.77. The method was found to be precise, and the relative standard deviation (RSD) was found to be between 0.28 and 1.92 with recoveries between 97.91 and 101.11. The method was found to be robust that resists deliberate changes in pH, flow rate, and mobile-phase percentages. It was successfully applied for the analysis of PDSs. The proposed method could be very useful for the quality control of the 10 structurally related AAs during their synthesis and for testing raw materials and pharmaceutical preparations.

Histone post-translational modifications (PTMs) alter chromatin dynamics and contribute to the regulation of gene expression in health and disease, yet mass spectrometry-based histone PTM analysis remains constrained by inefficient sample preparation workflows. Here, we develop RIPUP (Rapid Identification of histone PTMs in Underivatized Peptides), a streamlined multi-protease workflow that reduces sample preparation to hours while improving PTM coverage and quantitative accuracy. Systematic evaluation of Arg-C Ultra and a recombinant (r)-Chymotrypsin protease under varied conditions, including standard derivatization with propionic anhydride and tandem mass tag (TMT) labeling, demonstrated that Arg-C Ultra with TMT labeling achieves a detection of total PTM that exceeds Trypsin-based approaches. Using the HiP-Frag computational framework for unrestrictive PTM identification, we discovered that TMT's tertiary amine provides charge compensation that rescues the ionization of negatively charged acylations revealing 58 succinylation and 31 glutarylation sites - a 'dark epigenome' largely undetected by propionylation-based methods. Complementary digestion with Arg-C Ultra and r-Chymotrypsin provides orthogonal sequence coverage, enabling detection of PTMs in H2A variants, linker histones, and regions poorly represented by arginine-specific cleavage alone. In HEK293T cells treated with the pan-sirtuin inhibitor nicotinamide, RIPUP quantified 112 statistically significant peptidoforms (adj p < 0.05), predominantly increasing with NAM dose (88 up, 24 down). Application of RIPUP to frozen-thawed rat hippocampal sections within a 3-hour workflow identified >200 PTMs including H3 K27/K36/K37 methylation, H4 N-terminal acetylation patterns, and H2A K118/K119 ubiquitination. This rapid, high-efficiency platform enables timely discovery of epigenetic mechanisms and accelerates the path from PTM identification to therapeutic target validation. ### Competing Interest Statement The authors have declared no competing interest. National Institute on Alcohol Abuse and Alcoholism, T32 AA007456, AA013498, P60 AA006420, AA017447, AA029841, AA021491 Paul and Cleo Schimmel Endowed Chair

Histone post-translational modifications (PTMs) alter chromatin dynamics and contribute to the regulation of gene expression in health and disease. Mass spectrometry-based analysis is the gold-standard for histone PTM analysis, but it remains constrained by inefficient sample preparation workflows requiring multiple days. Here, we develop RIPUP (Rapid Identification of histone PTMs in Underivatized Peptides), a streamlined multi-protease workflow that reduces sample preparation from days to hours while improving PTM coverage and quantitative accuracy. Through systematic evaluation of the Arg-C Ultra protease and a prototype recombinant (r)-Chymotrypsin protease under varied conditions, such as chemical derivatization using propionic anhydride and tandem mass tags (TMT), we demonstrated that Arg-C Ultra with TMT labeling achieves a detection of total PTM comparable to conventional Trypsin-based approaches. Using the HiP-Frag computational framework for unrestrictive PTM identification, we discovered that TMT's tertiary amine provides charge compensation that rescues the ionization of negatively charged acylations revealing, 50 succinylation and 27 glutarylation sites - a 'dark epigenome' largely undetected by propionylation-based methods. We demonstrated that complementary digestion with Arg-C Ultra and r-Chymotrypsin provides orthogonal sequence coverage, enabling detection of PTMs in H2A variants, linker histones, and regions poorly represented by arginine-specific cleavage alone. Application of RIPUP to frozen-thawed rat hippocampal sections within a 3-hour workflow identifies >200 PTMs including biologically critical PTM sites H3 K27/K36/K37 methylation, H4 N-terminal acetylation patterns, and H2A ubiquitination at K118/K119. This rapid, high-efficiency platform enables timely discovery of epigenetic mechanisms and accelerates the path from PTM identification to therapeutic target validation.

Chiral analysis of amino acid enantiomers is essential due to their frequently divergent biological activities. However, ion mobility spectrometry–mass spectrometry (IMS–MS) cannot directly resolve underivatized amino acid enantiomers due to their identical physicochemical properties. This study developed a derivatization-based analytical method utilizing (S)-N-(4-nitrophenoxycarbonyl) phenylalanine methoxyethyl ester ((S)-NIFE) as a chiral derivatization agent to achieve the stereoselective modification of amino acids. The resulting diastereomers form metal ion adducts, which enhance collision cross section differences and enable separation via trapped ion mobility spectrometry and time-of-flight mass spectrometry (TIMS–MS). The differential impact of alkali metal ion adduction (Na+, K+, Rb+, and Cs+) on chiral separation was investigated, revealing that Na+ adducts provide optimal enantioselective recognition (average resolution, Rpp = 2.02). The established method achieved separation for 30 chiral amino acids, demonstrating faster speed, broader coverage, and superior resolution compared with existing approaches. When applied to rat blood samples from chlorfenapyr-induced toxicity models, the method successfully detected d-Arg, d-Ile, d-Leu, d-Phe, and d-Met, confirming its practical utility in toxicological diagnostics and biomarker discovery.

We report on a newly developed method for compound-specific isotopic analysis (CSIA) of carbon (δ13C) and nitrogen (δ15N) in underivatized pyrimidine (cytosine, uracil, and thymine) and purine (adenin...

We report on a newly developed method for compound-specific isotopic analysis (CSIA) of carbon (δ13C) and nitrogen (δ15N) in underivatized pyrimidine (cytosine, uracil, and thymine) and purine (adenine, guanine, hypoxanthine, and xanthine) nucleobases. The nucleobases are isolated by multistep purification using high-performance liquid chromatography (HPLC). HPLC separation and its wet chemical pretreatments were optimized to isolate these nucleobases without the need for chemical derivatization. Subsequently, the carbon and nitrogen isotopic compositions of these underivatized nucleobases were analyzed by nanoscale elemental analysis/isotope-ratio mass spectrometry (nano-EA/IRMS). The δ13C and δ15N measurements of isolated standard nucleobases indicated minimal isotopic fractionation during the two-step HPLC purification. This HPLC × nano-EA/IMS methodology was utilized to determine the isotopic composition of nucleobases in spinach (δ13C: −27.0 to −19.8‰, δ15N: +2.8 to +6.7‰) and coastal sediment (δ13C: −14.9 to −9.2 ‰, δ15N: +4.5 to +9.0‰) samples. The CSIA of nucleobases can provide insight into how living organisms modulate their biochemical pathways in response to environmental and metabolic variations.

Polysaccharides are essential resources for all organisms. Despite the increasing popularity and sophistication of biological macromolecule research, analytical approaches for polysaccharides are stil...

We describe the simultaneous quantification of 14 underivatized amino acids (AAs) in various matrices using ion-pairing liquid chromatography–tandem m…

Free amino acids (AAs) and their metabolites are important building blocks, energy sources and signa

Free amino acids (AAs) and their metabolites are important building blocks, energy sources and signa

Analyst, 2026, Accepted Manuscript DOI: 10.1039/D5AN01066G, PaperYujia Ying, Jiangang Chen, Shaoping Li, Jing Zhao Polysaccharides are essential resources for all organisms. Despite the increasing popularity and sophistication of biological macromolecule research, analytical approaches for polysaccharides are still urgently required due to the complex heterogeneity... The content of this RSS Feed (c) The Royal Society of Chemistry

Publication date: Available online 12 November 2025 Source: Journal of Chromatography A Author(s): Dong Wook Seok, Seohyun Choi, Ji-Seon Jeong

Studies on glycans of glycoproteins are hampered by the lack of standards that reflect the wide diversity in structure typically observed. To this end we have exploited a large library of N-glycan standards comprised of a unique collection of 226 structures including oligomannose, hybrid, and complex-type. We generated a method employing porous graphitized carbon (PGC) and liquid chromatography mass spectrometry (PGC-LC-MS), which can provide a high degree of resolution of underivatized N-glycan structures. Chromatogram libraries arising from these studies include retention time data, diagnostic fragments, and validated structural assignments, providing a robust platform for both targeted and discovery-based glycomics. We refer to this as an N-glycopedia, the first type of resource in which researchers can compare this collective data to N-glycans under study and overcome the limitations of only having compositional data and predicted structures. The technology is easily expandable to include additional N-glycans as new standards become available.