Search Results (301)

Tetrastigma hemsleyanum Diels et Gilg is a high-value edible and medicinal homologous plant, routinely grown under conventional field or greenhouse production systems across Asia. However, mislabeling of conventional products as the rarer (and more expensive) wild version may occur for financial gain. In this study, stable isotopes (δ¹³C, δ¹⁵N, δ²H, and δ¹⁸O) and metal contents (Cr, Cu, Ni, As, Cd, Pb) were used to characterize plant tissues (tuber root, stem, leaf) and corresponding soils originating from simulated-wild-cultivated (WC) and greenhouse-cultivated (GC) pot trials using the same soil. Carbon and nitrogen isotopes served as key indicators for distinguishing GC and WC products. Specifically, δ¹³C values of GC plant tissues were 1.4 to 2.4‰ more positive than those of WC plant tissues (p < 0.05), and δ¹⁵N values in GC tissues were 2.7 to 4.6‰ more positive than δ¹⁵N in WC tissues (p < 0.01). Lower δ¹⁵N values observed in WC products indicate slower nitrogen turnover compared with GC products. Soil metal concentrations had significant differences between the two cultivation systems, but only limited effects on metal bioconcentration factors (BCFs) and translocation factors (TFs) in T. hemsleyanum tissues. Pb and Cd concentrations in root tissues had large differences between cultivation systems, and carbon dynamics in GC plants were more negatively affected by Pb levels in soils. These findings provide the first investigation of T. hemsleyanum grown under different cultivation practices and establish a scientific basis for distinguishing other wild or simulated-wild labeled food and medicinal plant products from conventionally grown products in future studies. Full article

8-Hydroxy-dihydroergotamine is the major human metabolite of the anti-migraine drug dihydroergotamine and is required, along with a stable isotope-labelled derivative, to aid metabolic studies. An efficient, scalable synthesis of the unlabelled compound is described via the coupling of dihydrolysergic acid to the tricyclic amino compound (2R,5S,8R,10aS,10bS)-2-amino-5-benzyl-10b-hydroxy-8-methoxy-2-methyltetrahydro-8H-oxazolo[3,2-a]pyrrolo[2,1-c]pyrazine-3,6(2H,5H)-dione. The tricycle was obtained by a convergent synthesis combining precursors from suitably protected L-glutamic acid and L-phenylalanine, and 2-bromo-2-methylmalonic acid. For the labelled molecule, the tricyclic precursor contained a pentadeutero benzyl group derived from [2,3,4,5,6-²H₅]L-phenylalanine. Considerable experimentation was required to achieve optimal activation of dihydrolysergic acid for efficient amide formation with the tricycle’s amino function affording 8-methoxy-dihydroergotamine. The stereochemical integrity of an intermediate in this synthesis, ethyl (2R,5S,8R,10aS)-5-benzyl-10b-hydroxy-8-methoxy-2-methyl-3,6-dioxooctahydro-8H-oxazolo[3,2-a]pyrrolo[2,1-c]pyrazine-2-carboxylate, was validated by crystal structure analysis. Acid-catalysed hydrolysis of 8-methoxy-dihydroergotamine gave 8-hydroxy-dihydroergotamine. Pentadeuterated 8-hydroxy-dihydroergotamine was obtained in an analogous manner from [2,3,4,5,6-²H₅]L-phenylalanine. Both 8-hydroxy-dihydroergotamine and its ²H₅-derivative were obtained as an equilibrating mixture of C-8 epimers (diastereomers), with the major isomer having (R)-configuration according to ¹H NMR analysis. The syntheses described enable the routine synthesis of 50–100 mg quantities of each target molecule. Full article

Alterations in the ratio of oxidized and reduced nicotinamide adenine dinucleotide (NAD⁺/NADH) reflect the intracellular redox state and have been implicated in a broad spectrum of pathological conditions, including neurogenetic disorders, heart failure, and liver diseases. In the present study, we demonstrate the selective detection of probe-derived NAD⁺ and NADH signals in mouse liver extracts by means of a triple-resonance nuclear magnetic resonance (NMR) spectroscopy technique. We prepared ¹³C/¹⁵N-enriched nicotinamide riboside (¹³C/¹⁵N-NR), which undergoes enzymatic conversion to NAD⁺ and NADH in the liver, and detected these labeled metabolites by ¹H–{¹³C–¹⁵N} triple-resonance NMR measurements. This study provides a methodological proof-of-concept for the selective detection of NAD-related signals derived from a stable-isotope labeled probe in mouse liver extracts. Full article

Background: Untargeted proteomics enables quantitative host cell protein (HCP) determination in biotherapeutics, yet no workflow has been validated under ICH Q2(R2) for regulated quality control. Methods: A prospective total-error (TE) validation of label-free ddaPASEF proteomics was performed. A stable isotope-labeled whole-proteome standard was spiked into NISTmAb at seven levels (20–80 ng) and analyzed in four independent assays (198 injections), supporting one-way random-effects ANOVA with Welch–Satterthwaite adjustment. Peptide-level identification error was evaluated by dual entrapment. Results: Empirical false-discovery proportions were below 1% at q = 0.01. Weighted least-squares regression (R² = 0.993) confirmed stable proportional compression with 81–85% recovery. Repeatability dominated the variance structure (median CV 2.7%); intermediate precision SD ranged from 0.69% to 3.81%. Both 95% β-expectation and 95/95 content tolerance intervals were contained within ±30% at all levels, defining a validated range of 20–80 ng. Abundance-stratified TE profiling revealed concentration-dependent calibration heterogeneity, with stratum-specific intervals within ±35% defining an abundance-aware LLOQ of 3.6 ppm (P95 = 3.87 ppm). Robustness under independent search software (FragPipe v24.0, CCC = 0.998) and cross-platform acquisition (Astral, CCC = 0.980) remained within ±30% limits. Conclusions: This constitutes the first prospective ICH Q2(R2)-aligned validation of untargeted proteomics for HCP quantification, with a transferable statistical framework for high-dimensional analytical methods. Full article

Targeted delivery systems offer a promising approach for selectively modulating cellular processes; yet the intracellular consequences of targeted nutrient delivery to trophoblast cells remain poorly defined. Here, we investigated a previously validated placenta-targeting peptide conjugated to liposomes encapsulating stable isotope-labelled L-arginine and L-lysine to examine cellular uptake and downstream molecular responses in a trophoblast-like cell model. Peptide-dependent uptake of fluorescently labelled liposomes was confirmed in BeWo cells, demonstrating selective internalisation compared with non-targeted controls. Encapsulation of isotope-labelled amino acids enabled direct quantification of intracellular delivery and incorporation into the cellular proteome using stable isotope labelling by amino acids in cell culture (SILAC). Quantitative proteomic analysis revealed coordinated changes in proteins associated with translation, metabolism, and nitric oxide synthase regulation following targeted liposomal uptake. Notably, V-type proton ATPase subunit G1 (ATP6V1G1) and large neutral amino acid transporter small subunit 1 (SLC7A5) showed increased incorporation of labelled amino acids and were independently validated by Western blotting. Together, these findings establish a proof-of-concept platform for targeted intracellular amino acid delivery to trophoblast-like cells and define the resulting proteomic responses. This work provides mechanistic insight into intracellular amino acid utilisation and a framework for future studies in placental cell biology. Full article

The origin of life is commonly discussed within two competing conceptual frameworks: the metabolism-first and information-first hypotheses. While each emphasizes a different defining property of early life, modern biochemistry reveals a fundamental interdependence between metabolic processes and genetic information transfer, leading to a persistent chicken-and-egg problem. In this study, we investigate a prebiotically plausible reaction system that enables the concurrent formation of molecular precursors associated with both frameworks. Under simulated Hadean hydrothermal conditions, acetylene, ammonia, cyanide, and carbon monoxide were reacted in aqueous solution in the presence of transition metal sulfides. Using gas chromatography–mass spectrometry combined with stable isotope labeling, we demonstrate the simultaneous formation of the nucleobase uracil and the amino acids alanine and aspartic acid. Isotopic incorporation patterns allow reconstruction of the underlying reaction pathways and confirm the contribution of all starting materials to product formation. While amino acids are produced continuously over the observed period in significantly higher yields than uracil, uracil formation exhibits a pronounced time-dependent maximum after three days. Variations in pH, reaction time, and metal sulfide catalysts modulate product yields but do not prevent the parallel emergence of both molecular classes. These findings support a scenario in which proto-metabolic chemistry and molecular precursors of genetic information could have arisen simultaneously within a shared geochemical setting. The results provide experimental support for a coupled origin of metabolism and transcriptional building blocks, offering a potential resolution to the dichotomy between metabolism-first and information-first models of early life. Full article

Background: Stable isotope-based analytical methods have brought about a significant transformation in the study of energy nutrient metabolism, enabling precise in vivo measurement of metabolic fluxes at systemic, tissue, and organ-specific levels in both healthy and diseased states. The regulation of these metabolic fluxes is governed by dynamic interactions between proteins, lipids, carbohydrates, and their precursors—such as glucose, fatty acids, and amino acids—as well as final metabolic products. Discussion: Advanced analytical technologies, including nuclear magnetic resonance (NMR) spectroscopy and mass spectrometry (MS), which can offer enhanced precision, have been developed for investigating nutrient metabolism and fluxes in humans, providing precise information on metabolic pathways. These techniques have primarily utilized stable isotopes, such as ²H, ¹³C, ¹⁵N, and ¹⁸O, which have largely replaced radioactive isotopes and are now central to metabolic research. These isotopes have been used to label glucose, fatty acids, or amino acids—the main biomolecular precursors—enabling detailed investigation at systemic, tissue, and organ-specific levels of carbohydrate, lipid, and protein metabolism, and revealing pathway alterations associated with diseases conditions, such as diabetes, non-alcoholic fatty liver disease, cardiovascular disorders, and cancer. The use of deuterium oxide (D₂O) has allowed for long-term metabolic studies, providing a cost-effective and less invasive means to monitor metabolic changes over days to months. Total daily energy expenditure can be measured in free living conditions by the doubly stable isotopes ²H- and ¹⁸O-labeled water method. Stable isotope tracing, combined with advanced imaging and modeling, has also been instrumental in assessing body composition, energy expenditure, and nutrient bioavailability. Collectively, these methods have expanded our understanding of human physiology and disease, supporting the development of novel diagnostic tools, the identification of new biomarkers, and the tailoring of nutritional and therapeutic interventions. Conclusions: This review aimed to provide an overview of the applications of stable isotopes for the study of energy nutrient metabolic pathways. The ongoing integration of stable isotope approaches with artificial intelligence, omics technologies, and miniaturized detection techniques could promise to further refine our understanding of human metabolism and drive advances in personalized medicine. Full article

Matrix effects (ME) during LC-ESI-MS analysis are a commonly acknowledged issue for a variety of matrices and analytes. Although sample preparation techniques are steadily evolving to reduce ME, the complexity and variability of the urine matrix remain a challenge, especially for multi-analyte methods. To investigate the extent of ME implications on method performance and quantification, we used stable isotope-labelled standards (SIL-IS) of 11 mycotoxins to evaluate the magnitude and variability of ME in urine samples from two cohorts: Bangladeshi adult women (n = 50) and Swedish children of both sexes (n = 340). Significant ME differences were observed between the two cohorts for eight of the 11 mycotoxins. Additionally, intra-cohort ME variability turned out to be very high with interquartile ranges (IQR) above 15% for 14 out of 22 analyte-cohort combinations. Maximum IQR values were observed for sterigmatocystin in the Bangladeshi cohort (318%), strongly impacting quantitative results obtained with matrix(-matched) calibration. Further experiments on a small German cohort of four subjects, each providing four to five urine samples, revealed high variability of ME within each individual. Factors influencing ME were investigated, showing little to no impact of sex and a moderate impact of age for some analytes in the Swedish cohort. Nonetheless, especially the more polar analytes, showing stronger signal suppression, demonstrated clear correlation of ME with density and creatinine concentration of the urine samples. As a result, urine samples with very high or low density or creatinine values require careful handling in regard to sensitivity or quantification errors when matrix(-matched) calibration without SIL-IS is applied. Full article

Microscopic green algae are active producers of beneficial compounds, particularly those containing nitrogen. However, the metabolism of nitrogen-containing compounds is diverse and depends on the conditions of the nitrogen source. As a result, the approach to studying the metabolism of nitrogen-containing compounds becomes more complicated. This work demonstrates the metabolic changes in the high-productive green algae Neochlorella semenenkoi IPPAS C-1210 under conditions of nitrogen starvation and subsequent reintake, using high-performance liquid chromatography–mass spectrometry (HPLC–MS) with ¹⁵N isotopic labeling. The presented results include semi-quantitative chromatography–mass spectrometric analysis for 17 amino acids, a metabolomic profile of over 40 isotopically labeled compounds, an assessment of metabolic flux via isotopic incorporation, and an analysis of cellular lipid composition under varying growth conditions. The findings indicate that this strain can utilize ammonium acetate as a nitrogen source, consuming nitrogen in the ammonium form. The degree of isotopic labeling in compounds often diverged significantly from their quantitative changes (concentrations and chromatographic peak areas), suggesting that isotopic analysis may offer advantages over purely quantitative analysis for biological systems. Furthermore, in vivo biological isotopic labeling is shown to assist in identifying compounds absent from standard mass spectrometric databases. Full article

Tobacco smoke exposure not only increases the risks of lung cancer and cardiovascular disease, but can be a stressor contributing to mental illness. It is important to clarify the relationship between chronic tobacco smoke exposure and mental stress from the perspective of disease prevention. We developed a simple and highly sensitive method for simultaneously analyzing nine biomarkers: nicotine and cotinine (tobacco smoke exposure markers); cortisol, testosterone, and dehydroepiandrosterone (stress-related markers); and serotonin, melatonin, dopamine, and oxytocin (relaxation-related markers). Biomarkers were extracted and concentrated by in-tube solid-phase microextraction with a Supel-Q PLOT capillary, followed by separation and detection within 7 min using liquid chromatography–tandem mass spectrometry on a Discovery HS F5 column. Calibration curves using stable isotope-labeled internal standards showed good linearity (0.005–100 ng mL⁻¹) with detection limits of 0.09–13.5 pg mL⁻¹. Intra-day and inter-day precision had relative standard deviations below 7.2% and 15.5% (n = 6), respectively, with recovery rates of 84.0–108.8%. The automated method requires only ultrafiltration of hair methanol extract, enabling non-invasive pg-level analysis using just a few milligrams of hair. Hair analysis reflects an association between chronic tobacco smoke exposure and stress. This method is effective for analyzing the relationship between long-term tobacco smoke exposure and chronic stress. Full article

Background: Host cell proteins (HCPs) are process-related impurities that must be monitored in biopharmaceutical products due to their potential impact on product quality and patient safety. Targeted LC–MS/MS approaches such as multiple reaction monitoring (MRM) enable protein-specific HCP quantification but are difficult to apply in highly multiplexed assays because of retention time (RT) variability across complex bioprocess matrices. Methods: Here, we show that conventional RT-scheduled MRM workflows lack transferability when applied to heterogeneous drug substances and process intermediates. Using a targeted assay comprising 240 peptides corresponding to 97 CHO-derived HCPs, RT shifts of several minutes resulted in truncated chromatographic peaks and peptide signal loss, even when wide scheduling windows were used. To overcome this limitation, a scout-triggered MRM (st-MRM) acquisition strategy based on event-driven monitoring was implemented. Results: This approach enabled robust peptide detection across diverse matrices within a single injection, without method re-optimization. Absolute quantification using stable isotope-labeled peptides spanned six orders of magnitude, with HCPs quantified down to 2.9 ppm in purified drug substances. Conclusion: Overall, st-MRM improves the robustness and transferability of highly multiplexed targeted proteomics workflows for HCP analysis. Full article

Hexoses, particularly glucose, are one of the most essential molecules for sustaining life; therefore, reliable methods for their analysis are very important. In our study, we present a qualitative and quantitative approach for analysing hexoses using MALDI IMS (Matrix-Assisted Laser Desorption/Ionization Mass Spectrometry Imaging) with betaine aldehyde derivatisation and a CHCA (α-Cyano-4-hydroxycinnamic acid) matrix in positive ionisation mode. In this study, we demonstrated betaine aldehyde derivatisation of glucose from dried droplets and explored the analysis of hexoses in brain and liver tissue slices. We assessed whether our method could distinguish between mannose, galactose, glucose, and fructose and optimised the preparation of a biomimetic calibration curve using stable-isotope labelled glucose for hexose analysis. For this purpose, we investigated the number of betaine aldehyde layers required to obtain a proper calibration curve; examined whether changes in the spray nozzle position during CHCA matrix deposition could facilitate analysis and investigated how storage conditions influenced the calibration curve analysis. Finally, we optimised the technique for liver and brain analysis and assessed variations in hexose levels between brain, liver, kidney, and spinal cord tissues from control and morphine-addicted animals. We hope that our biomimetic approach to creating the calibration curve will be helpful for quantitative analysis and aid in developing various quantitative methods for assessing endogenous substances. Full article

Background/Objectives: Therapeutic drug monitoring (TDM) of β-lactams (BL), BL/β-lactamase inhibitor (BLI) combinations (BL/BLIc), and of fosfomycin may play a key role in optimizing antimicrobial therapy and in preventing resistance development, especially when used by continuous infusion in critically ill or immunocompromised patients. Unfortunately, analytical methods for simultaneously quantifying multiple BL/BLIc in plasma are still lacking. Methods: The aim of this study was to develop and validate two rapid, sensitive, and accurate UPLC–qTOF–MS/MS methods for the simultaneous quantification of five novel β-lactam or β-lactam/β-lactamase inhibitor combinations (ceftolozane/tazobactam, ceftazidime/avibactam, meropenem/vaborbactam, cefiderocol, and ceftobiprole) along with fosfomycin. Methods: Human plasma samples were prepared by protein precipitation using methanol containing isotopically labeled internal standards. Chromatographic separation was achieved within 10–12 min using two Agilent Poroshell columns (EC-C18 and PFP) under positive and negative electrospray ionization modes. The method was validated according to the EMA guidelines by assessing selectivity, linearity, precision, accuracy, matrix effects, extraction recovery, and stability. Results: The methods exhibited excellent linearity (R² ≥ 0.998) across the calibration ranges for all of the analytes (1.56–500 µg/mL), with limits of quantification ranging from 1.56 to 15.62 µg/mL. Intra- and inter-day precision and accuracy were always within ±15%. Extraction recovery always exceeded 92%, and the matrix effects were effectively corrected through isotopic internal standards. No carry-over or isobaric interferences were observed. All the analytes were stable for up to five days at 4 °C, but the BL and BL/BLIc stability was affected by multiple freeze–thaw cycles. Conclusions: These UPLC-qTOF-MS/MS multi-analyte methods enabled a simultaneous, reliable quantification in plasma of five novel beta-lactams and of fosfomycin. Robustness, high throughput, and sensitivity make these multi-methods feasible for real-time TDM, supporting personalized antimicrobial dosing and improved therapeutic outcomes in patients with severe or multidrug-resistant infections. Full article

China, as the primary importer of Thailand’s high-quality rice (Oryza sativa L.), has an urgent need for effective origin discrimination methods between premium aromatic rice from China and Thailand to prevent origin mislabeling issues. In this study, stable isotope and elemental multivariate analysis combined with partial least squares discriminant analysis (PLS-DA) were used to build an origin traceability model for Chinese and Thai rice from different production areas. Multivariate analysis of variance revealed that Thai rice exhibited significantly higher δ¹³C (−26.4 ± 0.4‰) and δ¹⁸O (25.9 ± 1.1‰) values, but a significantly lower δ¹⁵N value (3.5 ± 0.8‰) compared to the three major producing regions of China. These differences are directly related to geographical and climatic factors such as latitude, precipitation, and temperature. A PLS-DA model demonstrated high performance in the classification of different Chinese indica rice and Thailand rice origins. Through cross-validation, the classification accuracy for the training set reached 97.3%. For the independent testing set, the classification accuracy was recorded to be 95.0%. Furthermore, external blind sample verification was conducted, and the classification accuracy achieved was 100%. Ca, K, Na, δ¹⁸O, Zn and δ²H were found to be important variables to discriminate between Chinese indica rice and Thai rice. Finally, for country of origin labelling claims, this rice study provides the basis for a suitable regulatory method to detect mislabeled Thai origin rice and prevent fraud. Full article

Honey adulteration has long been a nuisance in local and international trade. Sugar syrup addition and false labeling of botanical origin have created a challenge in identifying fraudulent honey supplies and products. Stable carbon isotope ratio analysis (SCIRA) has been widely employed in honey authentication. While it is effective in identifying the addition of C4 plant-derived sugars, it does not provide information related to honey’s botanical source. This research investigated the combination of SCIRA and melissopalynology to provide a more robust assessment of honey integrity and showed that PCA analysis of δ¹³C together with sugar profiles could further improve the decision involving addition of sugar syrups. A total of 34 beekeeper honey samples were analyzed from 7 provinces of Thailand with a focus on longan honey. Twenty-four samples passed the δ¹³C criteria, exhibiting δ¹³C of bulk honey ranging from −28.53 ± 0.19‰ to −22.89 ± 0.08‰ and δ¹³C of extracted protein ranging from −29.30 ± 0.07‰ to −22.76 ± 0.03‰. Pollen profiling further eliminated honey of questionable and multifloral origins, yielding only eight samples that passed both criteria of being monofloral and not being adulterated with C4-derived sugars. These included six samples of longan honey and two honey samples of other botanical origins, yielding an overall passing rate of 23.5%. Our study showed that by combining SCIRA and melissopalynology, a robust determination of honey integrity could be achieved. Full article