In vivo solid-phase microextraction (SPME) is a minimally invasive, non-exhaustive sample-preparation technique that facilitates the direct isolation of low molecular weight compounds from biological matrices in living systems. This technique is especially useful for the analysis of phytocannabinoids (PCs) in plant material, both for forensic purposes and for monitoring the PC content in growing Cannabis spp. plants. In contrast to traditional extraction techniques, in vivo SPME enables continuous tracking of the changes in the level of PCs during plant growth without the need for plant material collection. In this study, in vivo SPME utilizing biocompatible C18 probes and liquid-chromatography coupled to quadrupole time-of flight mass spectrometry (LC-Q-TOF-MS) is proposed as a novel strategy for the extraction and analysis of the acidic forms of five PCs in growing medicinal cannabis plants. The SPME method was optimized by testing various parameters, including the extraction phase (coating), extraction and desorption times, and the extraction temperature. The proposed method was validated with satisfactory analytical performance regarding linearity (10-3000 ng/mL), limits of quantification, and precision (relative standard deviations below 5.5 %). The proposed method was then successfully applied for the isolation of five acidic forms of PCs, which are main components of growing medicinal cannabis plants. As a proof-of-concept, SPME probes were statically inserted into the inflorescences of two varieties of Cannabis spp. plants (i.e., CBD-dominant and Δ9-THC-dominant) cultivated under controlled conditions for 30 min extraction of tetrahydrocannabinolic acid (Δ9-THCA), cannabidiolic acid (CBDA), cannabigerolic acid (CBGA), cannabiviarinic acid (CBVA), and tetrahydrocannabivarinic acid (THCVA). The results confirmed that the developed SPME-LC-Q-TOF-MS method is a precise and efficient tool that enables direct and rapid isolation and analysis of PCs under in vivo conditions. The proposed methodology is highly appealing option for monitoring the metabolic pathways and compositions of multiple PCs in medicinal cannabis at different stages of plant growth.

Myelin pathology in demyelinating diseases is accompanied by lipid remodeling that remains challenging to characterize at spatial level using traditional mass spectrometry. We developed an optimized AP-MALDI-Orbitrap MSI pipeline, incorporating sample preparation improvements and mass recalibration, to investigate lipid dynamics in the cuprizone (CPZ) mouse model of demyelination. Dual-modality, untargeted lipid profiling was performed to map spatially resolved lipid alterations during demyelination and spontaneous remyelination in two key brain areas of male mice: corpus callosum (CC) and cortex (Ctx), with lipid identifications benchmarked against 4D-LC-TIMS-MS/MS. Demyelinated regions were identified using Black Gold II staining. Using 1 ppm mass tolerance, we annotated 154 and 133 lipids at the sum-composition level in CC and Ctx, respectively, with 60% validated by LC-MS/MS. Spatial lipid profiling revealed CPZ-induced alterations in sphingolipids, sulfatides, and glycerophospholipids, supported by reanalysis of a published snRNA-seq dataset from a mouse CPZ model. Long-chain ceramides (Cer) and hexosylceramides (HexCer) were reduced in demyelinated regions, with partial, region-specific recovery during remyelination. Short-chain sulfatides (SHexCer), sphingomyelins (SM) and seminolipids transiently increased in the CC during demyelination, while long-chain sulfatides decreased in both CC and Ctx. Additionally, we observed demyelination-induced upregulation of polyunsaturated glycerophospholipids in CC and phosphatidylinositols (PI) in cortex. Lipid subclass changes emerged as reliable markers of both demyelination and remyelination in the mouse brain. Region-specific alterations in lipid metabolism provide new insights into the processes of de- and remyelination. Notably, remyelinated fibers have a distinct lipid profile compared to intact myelin, suggesting that lipid-based therapeutic strategies could improve myelin repair.

Molnupiravir, an orally administered prodrug of β-d-N4-hydroxycytidine (NHC), is incorporated into newly synthesized RNA by viral RNA-dependent RNA polymerase (RdRp). It is used for treatment of SARS-CoV-2 infections. Incorporation of NHC triphosphate into viral RNA inhibits replication of the virus, at least in part by introducing deleterious mutations. However, there is limited information on NHC incorporation into host RNA and reports on the risk of mutagenicity that molnupiravir/NHC pose to the host are conflicting. We used two liquid chromatography-mass spectrometry (LC-MS) methods to evaluate the incorporation of NHC into RNA and DNA of host Vero E6 cells in a SARS-CoV-2 infection model. To test this, host and viral RNA were degraded to their ribonucleosides, while host DNA was degraded to deoxyribonucleosides. Subsequently, nucleic acid constituents were analyzed by LC-MS, which offers specific, direct, and quantitative determination of incorporation. Our findings revealed concentration dependent NHC incorporation into host cell RNA in both infected and uninfected cell cultures, reaching a maximum of 1 in 7,093 bases. Analysis of host DNA revealed no presence of deoxy-N4-hydroxycytidine down to a detection limit of 1 in 133,000 bases. Our findings therefore suggest minimal to no NHC incorporation into host DNA, indicating a low probability of significant host cell mutagenicity associated with its use.

Phosphorylated nucleosides and their derivatives sit at the center of cellular energetics, signaling, and biosynthesis, yet they are largely invisible to routine reversed-phase metabolomics. Their phosphate groups chelate metal ions, producing severe peak tailing and chromatographic irreproducibility that consign them to the "dark metabolome." We report nucleotidomics, a workflow that recovers this phosphorylated fraction on standard reversed-phase LC-MS. Each sample is split in two; one aliquot is analyzed as-is, the other treated with a nucleoside digestion mix that strips enzyme-accessible phosphate groups, returning phosphorylated forms to their parent nucleosides. Subtracting the untreated signal from the treated one cancels the pre-existing free nucleoside pool common to both aliquots, leaving ∑P: the summed abundance of the phosphorylated forms of each nucleoside. ∑P is therefore an indirect, semi-quantitative readout — it flags phosphate-bearing pools but cannot resolve mono-, di-, and triphosphates. Seven-minute analytical cycles keep total instrument time practical despite the doubled injection count. In two gemcitabine-treated pancreatic ductal adenocarcinoma cell lines, the paired injections returned 1071 putatively annotated metabolites and 23 endogenous phosphorylated species surviving manual curation. The method recapitulated the drug's established pharmacology: extensive phosphorylation of gemcitabine, formation of the deaminated metabolite dFdU and its phosphates, and depletion of the deoxyadenosine and deoxyguanosine phosphate pools, consistent with ribonucleotide reductase inhibition. Untargeted analysis revealed deoxyinosine depletion, likely reflecting reduced deoxyadenosine pools. A high-sensitivity fragmentation protocol with extended ion accumulation (sniperMS2) yielded candidate identifications of trace species: free queuosine phosphates and endogenous 5′-methylthioinosine, to our knowledge not previously measured directly in human cells. ∑P alone gave tentative evidence for GDP-fucose phosphorylation. By rendering the nucleotide constituent of the dark metabolome accessible, nucleotidomics extends routine metabolomic workflows to the cellular nucleotidome without the need for specialized chromatography.

Delayed cerebral ischemia (DCI) affects approximately 30% of aneurysmal subarachnoid hemorrhage (aSAH) survivors and is a leading cause of morbidity and mortality post-aSAH. Current risk assessment relies on radiographic severity scores that stratify populations based on hemorrhage characteristics but cannot predict DCI onset for individual patients. No plasma-based biomarkers for DCI prediction currently exist. We used liquid chromatography – mass spectrometry to perform plasma metabolomics in 60 aSAH patients (50% female, 30 DCI, 30 No-DCI) with longitudinal sampling until 7 days post-aSAH. Samples were retrospectively aligned to individual DCI onset times per patient, isolating 24–48 h and 48–72 h pre-event windows. Arginine emerged as the sole FDR-significant metabolite 24–48 h before DCI (q = 0.022, 39% depletion) and ranked #1 of 2,022 metabolites across three statistical methods (Student’s t-test, Welch’s t-test, Mann-Whitney U test, p = 10⁻⁵ to 10⁻⁴). Zero metabolites achieved FDR significance at 48–72 h, confirming temporal specificity. Independent logistic regression validated predictive performance (odds ratio 0.22, p = 0.001, AUC 0.808, 95% CI: 0.647–0.945), outperforming modified Fisher radiographic score (AUC 0.718, 95% CI: 0.593–0.830). The combination model of arginine and modified Fisher score performed best with AUC 0.856 (95% CI: 0.733–0.955), with arginine as the dominant factor. Mechanistic investigation revealed FDR-significant depletion of arginine/ornithine (q = 0.011) and glutamine/glutamate ratios (q = 0.011), consistent with increased arginase activity competing with nitric oxide synthesis. Plasma arginine represents a candidate single-molecule biomarker for DCI with a clinically actionable pre-event detection window, pending prospective validation. Temporal alignment to individual DCI onsets enabled detection of pre-event metabolic changes 24–48 h before symptom onset.

Thymidylate synthase (TS) is an enzyme responsible for the conversion of deoxyuridine monophosphate (dUMP) to deoxythymidine monophosphate (dTMP), with the co-substrate 5,10-methylenetetrahydrofolate (5,10-CH2-THF) as the methyl donor. TS is the only enzyme capable of de novo biosynthesis of dTMP in humans, a nucleotide crucial for DNA synthesis and therefore cell proliferation and survival. As such, TS is a major drug target in chemotherapy by compounds such as 5-fluorouracil. Due to the clinical and physiological importance of TS, the ability to accurately assay its activity is crucial. Several assays have been developed for this purpose, relying on spectrophotometry or radioisotope labeling methods. In this study, we have developed a liquid chromatography - mass spectrometry-based method for assessing TS activity by direct and specific measurement of the reaction product, dTMP. The assay was tested on mouse liver homogenates. We noted that excessive 5,10-CH2-THF concentration (400 mu M) led to substrate inhibition and therefore 200 mu M was used. The activity assayed at 1 mu M dUMP was linear with protein content and time (up to 60 min) and was 0.56 +/- 0.12 pmol/mg protein/min, in line with previously reported values. Additionally, by using a high mass resolution Orbitrap instrument side reactions were monitored, revealing major changes in folate pools and nucleotide metabolism. These findings highlight the value of the developed TS assay for routine TS activity monitoring in complex matrixes such as clinical material.