Abstract: This study aims to establish a detection method based on enzymatic hydrolysis combined with high-performance liquid chromatography (HPLC) technology for the quantitative analysis of yeast β-glucan content in composite yeast cultures. The method employs exo-1,3-β-glucanase plus β-glucosidase (A1 enzyme) to enzymatically hydrolyze total glucans in the sample. Amyloglucosidase plus invertase (B1 enzyme) along with trehalase (B2 enzyme) are used to enzymatically hydrolyze α-glucans in the sample, and purified lichenase (C1 enzyme) and β-glucosidase (C2 enzyme) to hydrolyze the cereal β-glucans in the sample. The glucose produced after hydrolysis is subjected to PMP derivatization and detected by HPLC. By applying the formula, the results of the three types of glucan content were obtained, namely X_A, X_B, and X_C. Ultimately, the content of yeast β-glucan in the composite yeast culture was calculated as X_A - X_B - X_C. The experimental results showed that glucose exhibited good linearity within the concentration range of 1-400 μg/mL, with a correlation range of 0.99999, a detection limit of 0.084 mg/L, a quantification limit of 0.280 mg/L, an average spiked recovery rate of 80.857 % to 105.456 %, and an RSD of less than 5 %. These data indicate that the method established in this study has high precision and good feasibility, making it suitable for the detection and analysis of yeast β-glucan in composite yeast cultures.

Abstract: A sensitive analytical method with a run time of 17 min is presented to detect and quantify free and combined amino acids (FAA/CAA) and nucleobases in complex marine matrices. The method combines hydrophilic interaction chromatography (HILIC) with quadrupole time-of-flight mass spectrometry (Q-TOF-MS). Retention time interday relative standard deviation (RSD) was 0.09–7.1%; peak area RSD was <6%; and individual limits of quantification (LOQs) ranged between 0.5 and 5 μg/L. The optimized solid-phase extraction (SPE) desalting protocol for salt water achieved a recovery of more than 50%, except for threonine (30%) and uracil (15%). Despite lower recoveries, compounds were consistently and reliably detected in ambient samples. Recovery RSD was ≤10% for most analytes, with uracil (23%) and cystine (13%) as exceptions. Hydrolysis in polypropylene vials to quantify CAA yielded recoveries between 61 and 102%. Matrix effects were evaluated via standard addition and isotope-labeled standards, revealing ion suppression/enhancement in desalted seawater samples, thus confirming the need for matrix-matched calibration. For aerosol particle extracts, no desalting procedure was necessary. The method was applied to polar-region samples, yielding atmospheric concentrations that ranged between 0.5 pg/m3 for adenine and 3.5 ng/m3 for glutamine. In seawater samples, concentrations ranged between 1.3 μg/L for glycine and 48 μg/L for uracil. Altogether, the optimized method enables reliable analysis, without the need for a derivatization step. Thus, it is allowing for the sensitive determination of amino acids and nucleobases that are challenging to analyze and rarely reported in environmental studies to date.

Abstract: Abstract. In this study, we present two optimized analytical methods for the quantification of molecular markers to attribute the contribution of various Volatile Organic Compound (VOC) oxidation products to Secondary Organic Aerosol (SOA). Those involve Ultrahigh Performance Liquid Chromatography Electrospray Ionization coupled to Ion Mobility Time-of-Flight Mass Spectrometry (UPLC/ESI-IMS-QTOFMS) and Gas Chromatography Mass Spectrometry (GC-MS). Liquid extraction was performed for both techniques, with an extra derivatization step with N,O-Bis(trimethylsilyl)trifluoroacetamide (BSTFA) containing 1 % trimethylchlorosilane (TMCS) for GC-MS analysis, enhancing the compound detection capacity. Between the two techniques, 23 biogenic and anthropogenic markers were identified, with five common species detected. Recoveries between 40 % and 170 % were observed for nitro-containing compounds and between 70 % and 140 % for aromatic and non-aromatic acids except for 3-methyl-1,2,3-butanetricarboxylic acid. Limits of detection <5 ng were observed by UPLC/ESI-IMS-QTOFMS analysis for 4-nitrophenol and 2-methyl-4-nitrophenol, while GC-MS (with BSTFA derivatization) analysis allowed better detection of lower mass compounds (for example limit of detection for 2-methylerythritol was 0.10 ng). While UPLC/ESI-IMS-QTOFMS allows for the analysis of high molecular weight compounds at high resolution and sensitivity, GC-MS analysis focuses on compounds of lower mass and higher polarity, together, these complementary methods provide a comprehensive tool for the quantification of organic markers arising from the airborne transformation of compounds of both biogenic and anthropogenic origins.

Abstract: This study presents an automated derivatization protocol utilizing 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate (AQC) for enantioselective amino acid analysis of peptides synthesized via liquid phase peptide synthesis, as exemplified by neurotensin. The primary aim was to enhance operational efficiency to manage the derivatization of large sample sets and reduce human error in routine enantioselective amino acid analysis of peptide therapeutics. The chromatographic method based on Chiralpak QN-AX demonstrated enantio- and chemoselectivity for all proteinogenic amino acids (except D-Leu/D-Ile and Glu/pGlu), with quantitative analysis achieved by HPLC-ESI-MS/MS with MRM acquisition through external calibration using stable isotope-labeled internal standards. The goal was to test for racemization of amino acids during peptide synthesis and process optimization, respectively. The results confirmed varying susceptibility to racemization among amino acids during peptide synthesis and cleavage of protection groups, with specific amino acids exhibiting higher levels of D-enantiomer formation. The developed protocol effectively assessed the amino acid composition and stereointegrity of the liquid phase synthesized neurotensin. This research and application highlights the critical role of automation in optimizing peptide analysis workflows and sets the foundation for future improvements in peptide synthesis and chromatographic conditions to enhance specificity, particularly for challenging amino acid pairs. Ultimately, the findings contribute to advancing laboratory practices in peptide chemistry, ensuring the quality and efficacy of peptide-based therapeutics.

Abstract: Glyphosate and its primary metabolite aminomethylphosphonic acid (AMPA) are herbicide residues of significant concern in agricultural products like canola oilseeds. This study developed and validated a sensitive UHPLC-MS/MS method with FMOC-Cl derivatization to quantify glyphosate and AMPA in canola oilseeds. The method demonstrated high linearity, with R² values of 0.9997 for glyphosate and 0.9996 for AMPA. Recovery rates were 83.09% for glyphosate and 82.28% for AMPA, indicating good accuracy and precision. The limits of detection (LOD) were 0.0009 mg/kg for glyphosate and 0.0004 mg/kg for AMPA, and the limits of quantification (LOQ) were 0.0031 mg/kg and 0.0014 mg/kg, respectively. Application to real samples from Dezful and Kermanshah showed higher seed (canola) residues in Kermanshah for both glyphosate and AMPA, whereas chaff residues were higher in Dezful. The method complies with international regulatory standards, including those of Codex Alimentarius and the European Union, providing a reliable tool for food safety monitoring and regulatory compliance. • FMOC-Cl derivatization enabled sensitive LC–MS/MS detection of glyphosate and AMPA. • Matrix-matched calibration ensured accuracy in oilseed (canola) matrices. • Method validated according to SANTE/11312/2021 guidelines. • Oilseed-specific challenges (lipids, co-extractives) were successfully managed. • Approach supports reliable monitoring of glyphosate/AMPA in edible oils and seeds.

Abstract: Herein, we report a selective synthesis of 3,4-dihydronaphthalene derivatives from o-quinodimethane (o-QDM) intermediates by gold(I). This method is achieved through [(BINAP)₂Au₂(BAr^F₄)₂]-catalyzed reactions of o-alkynylarylaldehydes with alkenes following 1,5-H migration. The reaction features a broad substrate scope and excellent functional group tolerance. Notably, the catalytic system was highly efficient at 0.1 mol % loading for most reactions, which could be reduced to 0.05 mol % in gram-scale reactions while maintaining the yield. More significantly, the reaction enables late-stage modification of complex molecules, providing a new strategy for drug derivatization and efficient synthesis of bioactive trioxifene.

Abstract: Dipeptides have shown great potential in improving the production performance of livestock such as cattle, sheep and goats. The ruminal fluid, a complex matrix sample, is a good research object to investigate the healthy status of ruminants and to discover the biomarkers. A pre-column derivatization with dansyl chloride method was established for accurate quantification of 47 dipeptides by liquid chromatography (LC) triple quadrupole linear ion traps tandem mass spectrometry. An optimized LC gradient ensured the separation of Dns-dipeptides on Kinetic XB-C 18 column by the MRM mode. The ruminal fluid was treated with acetonitrile, and then directly derivatization with dansyl chloride under the optimized pretreatment conditions. The linear correlation coefficients were all above 0.99 in the linear range, the detection limits and the limits of quantitation of Dns-dipeptides ranged within 0.03–6.06 ng/mL and 0.09–20.0 ng/mL, respectively. The accuracy and precision were both less than 15.0 %. The average recovery rate of most analytes was between 80.0 % and 122.0 %. Most analytes were stable in 4 °C for three days. Twenty-five dipeptides were detected in the ruminal and abomasal fluid samples collected from the goats fed with corn and soybean-based diet (CS) or mulberry silage (MS) by the method developed. Dns-Gly-Pro, Dns-Gly-Val and Dns-Phe-Ala were the most abundant in the ruminal fluid, while concentrations of Dns-Phe-Ala, Dns-H-Tyr-Leu and Dns-Leu-Leu were the greatest in the abomasal fluid. Concentrations of five dipeptides, including Dns-Trp-Glu-OH, Dns-L-Ala-L-Leu, Dns-Leu-Leu, Dns-Tyr-Glu-OH and Dns-Val-Tyr, in the ruminal fluid of MS group were consistently decreased ( P < 0.05). However, Dns-H-Pro-Phe-OH level in the abomasal fluid of MS group was increased ( P < 0.05). The development of this quantitative detection method will greatly facilitate the understanding of the mechanism of dipeptides metabolism and circulation in the gut of livestock. • The LC-ESI-MS/MS method for simultaneous determination of forty-seven kinds of dipeptides in ruminal fluid with pre-column derivation was first reported. • Developed method can be used to determine the dipeptide profile in abomasal fluid.

Abstract: Abstract Purpose The aim of this work is to investigate whether precyclization of γ-hydroxybutyric acid (GABA) allows for increasing its gas chromatography (GC) signal, and if so, is it a more effective way to increase the signal of this compound than its silylation or methylation? Methods Gas chromatography-mass spectrometry (GC–MS) and GC with flame ionization detection (GC-FID) response to GHBA before and after silylation, methylation, and cyclization were compared. The impact of injector temperature on GHBA and γ-butyrolactone (GBL) signals was assessed. Fourier transformed infra-red spectroscopy was used to examine the formation of macromolecular derivatives in the injector. Results GHBA shows a lower GC signal than GBL due to partial polycondensation into a non-volatile polyester in the injector. Validation data were established for GHBA after each derivatization. Silylation and methylation reduced the limit of detection (LOD) by approximately 1.5- and 1.3-fold, respectively, whereas pre-cyclization led to at least a 4.6-fold decrease in LOD. Conclusions The present study elucidates the reasons behind the low GHBA signal observed in GC analysis and, consequently, supports the recommendation to perform pre-cyclization of this compound prior to analysis. Furthermore, the findings demonstrate that although signal enhancement of GHBA can be achieved through silylation or methylation, the most substantial increase is observed following its cyclization during sample preparation. The proposed in this paper cyclization procedure is both remarkably simple and highly effective, allowing for reliable quantification of this hydroxycarboxylic acid in a variety of matrices, including plasma, urine, wine, beer, and orange juice.

Abstract: The stability of collagen, the most abundant protein in humans and many animals, is related to the hydroxylation of L-proline, a post-translational modification occurring at carbon 3 and 4 on its pyrrolidine ring. Collagens of different origins have shown different proline hydroxylation levels, making hydroxyprolines useful biomarkers in structure characterizations. The presence of two chiral carbon atoms, 3-hydroxyproline and 4-hydroxyproline, results in eight stereoisomers (four pairs of enantiomers) whose quantitation in collagen hydrolysates requires enantioselective analytical methods. Capillary electrophoresis was applied for the separation and quantitation of the eight stereoisomers of 3- and 4-hydroxyproline and D,L-proline in collagen hydrolysates. The developed method is based on the derivatization with the chiral reagent (R)-(-)-4-(3-Isothiocyanatopyrrolidin-yl)-7-nitro-2,1,3-benzoxadiazole, enabling the use of a light-emitting diode-induced fluorescence detector for high sensitivity. The separation of the considered compounds was accomplished in less than 10 min, using a 500 mM acetate buffer pH 3.5 supplemented with 5 mM of heptakis(2,6-di-O-methyl)-β-cyclodextrin as the chiral selector. The method was fully validated and showed the adequate sensitivity for the application to samples of collagen hydrolysates. The analysis of samples extracted from chicken Pectoralis major muscles affected by growth-related myopathies showed different stereoisomer patterns compared to those from the unaffected control samples.

Abstract: The Korean food composition database lacks reliable analytical data on the vitamin K1 (phylloquinone, PK) and vitamin K2 (menaquinone, MK) contents of commonly consumed eggs in Korea. This study aimed to determine the vitamin K content in eggs using solvent extraction and enzyme extraction, followed by HPLC analysis with post-column derivatization and fluorescence detection. To ensure the validity of the analytical method, key validation parameters, including accuracy, precision, detection and quantification limit, and linearity, were thoroughly evaluated. A total of 20 different types of eggs were analyzed for their PK and MK contents. PK content ranged from 0.11±0.001 μg/100 g to 1.36±0.01 μg/100 g. The vitamin K2 content ranged from 15.04±0.03 μg/100 g to 165.97±6.92 μg/100 g. The vitamin K content in fertilized eggs was significantly higher than in commercial eggs. The yolk of the egg is abundant in vitamin K but undetectable in the egg whites. This study provides reliable nutritional data on vitamin K, contributing to the development of an accurate and comprehensive Korean food composition database.

Abstract: Macrocycles are abundantly used by nature to enable cell‐permeable bioactive molecules. Synthetic non‐peptidic macrocycles are also increasingly considered as modalities for difficult‐to‐bind proteins but guidelines for macrocyclization are only beginning to emerge. Macrocycles are thought to constrain the available conformations but also to allow for residual flexibility, the latter being poorly understood. Here we show that even medium‐sized macrocycles display an unexpected high conformational plasticity, even when bound to their protein target. Minor modification of the linker region of macrocycles can shift the conformational ensemble to distinct conformational subclasses, each constituting distinct three‐dimensional scaffolds for further optimization. This led to several new ligands with improved affinity and beneficial physicochemical parameters for the FK506‐binding protein 51, a promising target for depression, obesity and chronic pain. Importantly, none of the beneficial modifications could have been identified by classical medicinal chemistry as they only work in the macrocyclic context. Our results show that macrocyclization can do more than keeping loose ends together but rather provide a platform for multiple series of macrocycles with distinct binding modes.

Abstract: ABSTRACT Unsaturated fatty acids are susceptible to lipid oxidation through autoxidation, photooxygenation or enzymatical oxidation. A characteristic feature of enzyme‐catalyzed oxidation is the high regio‐ and stereospecificity of the formed fatty acid hydroperoxides. In this study, we present a method to quantify enzymatic lipid oxidation through reducing hydroperoxy fatty acid methyl esters to hydroxy fatty acid methyl esters and derivatizing them with enantiopure (S)‐ibuprofen, allowing the resolution of the enantiomer pairs as diastereomers via achiral GC‐MS. After application to enantiopure reference fatty acids, the approach was applied to autoxidation products of linoleic acid, and the expected racemic mixtures of the 9‐ and 13‐hydroperoxide derived hydroxy fatty acids were detected. On the other hand, when linoleic acid was oxidized using soybean lipoxygenase, clear enantiomeric excess of the (13S) enantiomer could be detected, proving the applicability of this method to detect enzymatic oxidation through enantiomeric excess.

Abstract: Abstract Objectives A new candidate isotope dilution-liquid chromatography-tandem mass spectrometry (ID-LC-MS/MS)-based reference measurement procedure (RMP) has been developed for the accurate and precise quantification of 17β-estradiol (E2) in human serum and plasma covering a measurement range from 0.400 to 5,000 pg/mL (1.47–18,357 pmol/L). To address this broad range, two separate methods were created: a high sensitivity (HS) method for concentrations between 0.400 and 5.00 pg/mL (1.47–18.4 pmol/L) and a standard range (SR) method for concentrations between 5.00 and 5,000 pg/mL (18.4–18,357 pmol/L). Methods As the primary reference material, E2 (CRM 6004-a) from the National Metrology Institute of Japan was used to ensure traceability to the international system (SI). A two-dimensional heart-cut LC approach was utilized for LC-MS/MS analysis, employing a supported liquid extraction sample preparation protocol for the SR method and a liquid-liquid extraction protocol followed by derivatization for the HS method. Assay validation was conducted following current guidelines. Selectivity and specificity were assessed using spiked serum samples, while potential matrix effects were evaluated through a post-column infusion experiment and comparison of standard line slopes. Precision, accuracy, and trueness were determined using an extensive 5-day protocol. Standard measurement uncertainty was evaluated according to the Guide to the Expression of Uncertainty in Measurement (GUM), with three individual sample preparations performed on at least two different days. Equivalence with higher-order RMPs was demonstrated through participation in the CDC Steroid Hormones Standardization (HoSt) program. Results The RMP enabled the quantification of E2 within the range of 0.400–5,000 pg/mL (1.47–18,357 pmol/L), demonstrating no interference from structurally related compounds and no evidence of matrix effects. The relative mean bias of the SR method ranged from −2.4 to 1.9 % across all levels, including secondary reference materials and spiked samples, whereas the HS method exhibited a mean bias ranging from −3.0 to 2.9 %. Expanded measurement uncertainties (k=2) for target value assignment ranged from 1.7 to 4.4 % for the SR method and were found to be ≤6.1 % for the HS method. The method’s transferability was demonstrated in a comparison study at a second laboratory. Additionally, the candidate RMP exhibited excellent correlation and equivalence to JCTLM-listed RMPs through the CDC HoSt program. Conclusions In summary, the ID-LC-MS/MS-based RMP accurately quantifies E2. Its robust performance makes it suitable for standardizing routine assays and measuring individual patient samples, ensuring traceability.