Search Results (31)

Heterozygous mutations in the GBA1 gene, encoding the enzyme glucocerebrosidase (GCase), are major risk factors for Parkinson’s Disease (PD). Ambroxol, a small chaperone originally used as a mucolytic agent, has been shown to cross the blood–brain barrier, enhance GCase activity, and reduce α-synuclein levels, making it a promising therapeutic candidate for disease-modifying effects in GBA1-associated PD (GBA1-PD). This study aimed to develop a method to quantify ambroxol levels in human plasma and cerebrospinal fluid (CSF) using liquid chromatography–tandem mass spectrometry (LC-MS/MS). Ambroxol was determined by online solid-phase extraction (SPE), coupled with LC-MS/MS, by gradient elution on a monolithic column. Detection employed a 3200 QTRAP tandem mass spectrometer in the positive electrospray ionization mode. Calibration curves exhibited linearity across the analyzed ranges in both plasma and CSF. The recovery rate ranged from 106.7% to 113.5% in plasma and from 99.0% to 103.0% in CSF. No significant matrix effect was observed. Intra-day and inter-day precisions were below 11.8% in both matrices, and accuracy ranged from 89.9% to 103.1% in plasma and from 96.3% to 107.8% in CSF. We evaluated and confirmed the stability of the analyte in plasma and CSF across various storage conditions. The method was successfully validated according to European Medicine Agency (EMA) guidelines and its applicability was confirmed in the context of a multicenter, randomized, double-blind, placebo-controlled, phase II study, designed to monitor the ambroxol levels in the plasma and CSF of GBA1-PD. Full article

Background: Diacerein, a prodrug of Rhein, is commonly prescribed for the management of joint disorders, specifically osteoarthritis. This study aimed to develop and validate an LC-MS/MS method to quantify Rhein and its major metabolites, Rhein-G1 and Rhein-G2, in plasma samples. Method: An ACE C18 column was used for chromatographic separation with a mobile phase comprising ammonium acetate at a concentration of 1.0 mM and acetonitrile. Detection was achieved using a Sciex 4000 Q-Trap LC-MS/MS, operated in negative ion mode with multiple reaction monitoring (MRM). Results: The analytical results indicated that the lower limit of quantification (LLOQ) for Rhein and its glucuronides was 7.81 nM. Precision was consistently below 9.14%, while accuracy remained within the acceptable range of 80.1–104.2%. We also verified the method’s matrix effect recovery and stability variance, which were less than 12.60% and 10.37%, respectively. The pharmacokinetic study demonstrated that diacerein is swiftly metabolized into Rhein, and then Rhein subsequently undergoes glucuronidation, forming detectable concentrations of Rhein-G1 and Rhein-G2 in plasma. Conclusions: This new LC-MS/MS method proved to be both sensitive and selective, allowing for pharmacokinetic studies in rats. Full article

This study aimed to characterize carotenoid profiles and unravel the genetic mechanisms underlying flesh color variation in white and yellow-fleshed peaches, with a focus on the hybrid cultivar ‘ZY29’ derived from two white-fleshed parents (‘Yulu’ and ‘Hujing Honey Dew’). Using UPLC-APCI-MS/MS, we quantified carotenoids in the pericarp (exocarp) and flesh (mesocarp) of parental and hybrid fruits. Results showed that ‘ZY29’ accumulated significantly higher levels of β-carotene and lutein compared to its white-fleshed parents. Transcriptome analysis revealed upregulation of carotenoid biosynthesis genes (PSY, LCYB, and ZDS) and downregulation of the carotenoid cleavage gene CCD4 in ‘ZY29’, explaining enhanced carotenoid accumulation. Integrative metabolome-transcriptome analysis identified core regulatory networks associated with metabolic shifts, including transcription factors (MYB and WRKY). These findings provide novel insights into the molecular basis of yellow flesh formation in peaches, offering potential targets (PSY and LCYB) and metabolic markers (β-carotene and lutein) for breeding nutritionally enriched cultivars. These findings contribute to a better understanding of the genetic factors and parental regulatory mechanisms involved in the formation of yellow flesh color in peaches. Our results have important implications for breeding new peach varieties with desirable color and nutritional qualities and may provide valuable insights for future research in this area. Full article

The Italian Carciofo di Paestum (C. scolymus) PGI, an artichoke variety from the Campania region, was investigated for its potential to reuse by-products for food supplements. EtOH:H₂O 50:50 and 75:25 extracts of its leaves were analyzed for phenolic and flavonoid content and antioxidant activity (TEAC: 1.90 and 1.81 mM of Trolox; DPPH IC₅₀: 106.31 µg/mL and 128.21 µg/mL; FRAP: 1.68 and 1.58 mM FeSO₄/g extract). To further investigate the antioxidant potential, the ability of the two extracts to scavenge reactive species was assessed in Caco-2 cell cultures, showing a dose-dependent antioxidant capacity. To highlight metabolites responsible for the activity, LC-ESI/HRMSMS analysis was achieved, revealing 28 compounds (sesquiterpenes, megastigmanes, quinic acid and hydroxycinnamic acid derivatives, flavonoids, lignans, triterpenoid saponins, and polar fatty acids), of which structures were determined using 1D- and 2D-NMR analysis. In addition, quantitative determination of caffeoyl, dicaffeoyl, and quinic acid derivatives (CQAs) was performed through LC-ESI/QTrap/MS/MS, highlighting that the most abundant compound was 5-caffeoylquinic acid (6), with values of 9.310 and 7.603 mg/g extract in EtOH:H₂O (75:25) and EtOH:H₂O (50:50), respectively. The analysis showed that extracts were rich in bioactive compounds, suggesting their potential for development into antioxidant-based food supplements that may protect cells from oxidative stress and support overall wellness. Full article

Spinacia oleracea L. cultivar platypus leaves are identified as a functional food due to their nutrient composition which promotes health beyond basic nutrition. Considering the increasing use of food supplements, S. oleracea baby leaves have been extracted by maceration, solid–liquid dynamic extraction (SLDE)-Naviglio, and ultrasound-assisted extraction (UAE) using EtOH and EtOH:H₂O mixtures. The analysis of the extracts by using LC-ESI/HRMSMS revealed 42 compounds (flavonoids, polar lipid derivatives, and 20-hydroxyecdysone), along with primary metabolites, detected by NMR analysis. A principal component analysis (PCA) of LC-ESI/HRMS and NMR data was performed, revealing how 20-hydroxyecdysone and flavonoids, the specialized metabolites mainly responsible for the biological activity of S. oleracea leaves, occurred in the highest amount in the EtOH and EtOH:H₂O (70:30, v/v) extracts obtained by SLDE-Naviglio extraction. 20-hydroxyecdysone was also quantified in all the extracts via LC-ESI/QTrap/MS/MS using the Multiple Reaction Monitoring (MRM) method. The EtOH extracts obtained by SLDE-Naviglio and maceration showed the highest content (82.16 and 81.27 mg/g extract, respectively). The total phenolic content (118.35–206.60 mg GAE/g), the flavonoid content (10.90–41.05 mg rutin/g), and the Trolox Equivalent Antioxidant Capacity (TEAC) (1.63–2.05 mM) of the extracts were determined. The EtOH:H₂O (70:30, v/v) extract analyzed by using SLDE-Naviglio showed the highest phenolic and flavonoid content and radical scavenging activity. Full article

The request for skin-whitening agents and bioactive principles able to control hyperpigmentation disorders is continuously growing. Chamomile (Matricaria chamomilla) is used as a remedy for skin diseases, but little is known about the ability of Roman chamomile (Chamaemelum nobile) to act as a skin-whitening agent. With the aim to investigate antioxidant and lightening potential, fresh aerial parts of C. nobile were extracted by maceration, ultrasound-assisted extraction, and solid–liquid dynamic (SLDE-Naviglio) extraction using EtOH/H₂O mixtures. Moreover, 32 metabolites (flavonoids, sesquiterpenoids, amides, and polar fatty acids) were identified by liquid chromatography/mass spectrometry. Principal component analysis revealed how the extract EtOH/H₂O 50% (Naviglio and long maceration), along with the extract EtOH/H₂O 60% (maceration) were richest in flavonoids. All extracts were tested by TEAC and DPPH assays, and to determine their in vitro antioxidant activity, the DHR 123 probe–intracellular ROS assay in HaCaT cells, for some extracts, was performed. Moreover, their ability to exert a whitening effect was tested by analyzing their tyrosinase inhibitory activity. The quantitative determination of apigenin, known as a natural tyrosinase inhibitor, was performed by LC-ESI/QTrap/MS/MS using the multiple reaction monitoring (MRM) method. These results are promising for selecting an extraction method to obtain a sustainable product rich in bioactives. Full article

Assessment of dietary intake remains a large challenge in nutrition studies. The application of food intake biomarkers is a promising approach to complement widely used self-reported intake assessments and to improve accuracy. The development of metabolomics has enabled the discovery of many potential food intake biomarkers, but their applications are still limited. We aim to develop a semi-quantitative LC-MS/MS procedure to analyze a panel of plasma metabolites reflecting dietary exposure in a wide context. Our approach relies on a multi-analyte targeted LC-MS/MS method using a LC-QTRAP and commercially available reference compounds. A panel of 347 metabolites was selected, representing dietary intake (fruits and vegetables, coffee, tea, heat-treated food, wholegrain cereals, berries, cruciferous vegetables, and seafood) and key metabolites in the endogenous metabolism (fatty acids, amino acids, cholesterol metabolism, Krebs cycle, bile acids, and microbial metabolism) which are affected by specific diets, as well as lifestyle exposures, such as smoking and alcohol consumption. The application of this panel will help in assessing dietary exposures and their relationships to health outcomes. We will present the status of the work. Full article

In August 2020, the Eruca sativa cultivar “Rucola della Piana del Sele” obtained from the European Union the prestigious PGI (protected geographical indication) label, which certifies the uniqueness of its characteristics and increases its prestige both nationally and, above all, internationally. This plant is recognized as a product of excellence, with a unique flavor and unmistakable aroma. Therefore, since there are no methods to characterize the PGI product, a metabolomic approach was applied to characterize E. sativa grown in the Piana del Sele and different geographical areas. As E. sativa has very wide cultivation, this study sought to compare the metabolite profiles of rocket grown in Piana del Sele, Bergamo, and Brescia, as well as in Switzerland, making a comparison also with the metabolite profile of E. sativa grown spontaneously. To determine the best procedure to distinguish “Rucola della Piana del Sele” from the others, different extraction procedures were carried out using different solvents and fresh or freeze-dried plant matrices. The different extracts were analyzed by liquid chromatography coupled with high-resolution mass spectrometry experiments, using chemometric analyses to identify biomarker metabolites that characterize the PGI product. The LC-ESI-Q-Exactive-MS/MS profiles of methanol and hydroalcoholic extracts of different cultivars of E. sativa were found to be rich in bioactive compounds such as glucosinolates, glycosylated flavonoids, fatty acids, and lipids. The LCMS data were analyzed by principal component analysis (PCA); the score scatter plot shows significant separation among Eruca samples grown in different geographical areas. In detail, loading the scatter plot revealed Eruca grown in Piana del Sele to be richer than other cultivars in glycosylated quercetin 3,3′,4′-O-triglucoside (7), quercetin-3,4′-O-diglucoside-3′-O-(6-sinapoyl-glucoside) (10), and quercetin diglucoside (30). Finally, considering the biological interest in erucin, the myrosinase product of glucoerucin, the latter was quantified in the extracts by LC-ESI/QTrap/MS/MS using the multiple reaction monitoring (MRM) method; E. sativa from Piana del Sele showed the highest content of glucoerucin. Full article

A number of steroids, including glucocorticoids and sex hormones, have been associated with neurodegenerative and cardiovascular conditions common in aging populations. The application of liquid chromatography tandem mass spectrometry (LC-MS/MS) steroid analysis offers an opportunity to conduct simultaneous multiplex steroid analysis within a given sample. In this paper, we describe the application of an LC-MS/MS steroid analysis method for the assessment of reference ranges of steroids in human saliva samples (200 µL) collected from older adults (age 50 years and above) enrolled in a European cohort investigating the risk for Alzheimer’s dementia. Saliva samples were prepared using supported liquid extraction (SLE) along with a calibration curve and analysed using a Waters I-Class UPLC (Ultra Performance Liquid Chromatography) and a Sciex QTrap 6500+ mass spectrometer. Mass spectrometry parameters of steroids were optimised for each steroid and a method for the chromatographic separation of 19 steroids was developed. Lower limits of quantitation (LLOQs), linearity and other method criteria were assessed. In total, data from 125 participants (500 samples) were analysed and assessed for reference ranges (64 male, 61 female). A total of 19 steroids were detected in saliva within the range of the method. There were clear diurnal patterns in most of the steroid hormones detected. Sex differences were observed for androstenedione (A4), testosterone (T), cortisone (E) and aldosterone (Aldo). In the first sample of the day, dehydroepiandrosterone (DHEA) was significantly higher in healthy volunteers compared to those with Alzheimer’s disease biomarkers. This LC-MS/MS method is suitable for the analysis of 19 steroids in saliva in adults. Full article

Among the environmental factors, seasonality is the one which most affects the metabolome of a plant. Depending on the harvest season, the plant may have a variable content of certain metabolites and thus may have different biological properties. Foeniculum vulgare is an annual plant whose cultivation creates large amounts of waste rich in bioactive compounds. The present investigation was performed with the aim of determining the amount of biologically active compounds in F. vulgare wastes obtained from varieties of different seasonality. Ten polyphenolic compounds were quantified in the little stems and leaves of Tiziano, Pegaso, and Preludio cultivars by ultra performance liquid chromatography (UPLC) hyphenated to QTRAP mass spectrometry by using the MRM (multiple reaction monitoring) method. The antioxidant activity of hydroalcoholic extracts was then evaluated using TEAC and DPPH spectrophotometric assays, followed by a multivariate statistical analysis to determine the correlation between metabolite expression and antioxidant activity. The Preludio variety, grown in summer, showed a higher content of bioactive compounds, which guarantees it a better antioxidant power; kaempferol 3-O-glucuronide, quercetin 3-O-glucuronide, and quercetin 3-O-glucoside are the polyphenolic compounds that could be mainly responsible for the antioxidant effect of fennel. The PLS chemometric model, which correlated quantitative data obtained by a sensitive and selective LC-ESI-QTrap-MS/MS analysis of antioxidant activity, resulted in a selective tool to detect the compounds responsible for the activity shown by the extracts in chemical tests. Full article

Myrtle liqueur production generates high amounts of by-products that can be employed for the extraction of bioactive compounds. Bio-based, non-toxic and biodegradable solvents (ethyl acetate and 2-methyltetrahydrofuran), and a mechanical extraction were applied to myrtle seeds, by-products of the liqueur production, to extract oils rich in phenolic compounds. The oils obtained were characterized for yield, peroxide value (PV), lipid composition, and total phenolic concentration (TPC). The phenolic profile of the oils, determined by LC-MS, the antioxidant activity, and the oxidative stability were also analyzed. A validated UHPLC-ESI-QTRAP-MS/MS analytical method in multiple reaction monitoring (MRM) mode was applied to quantify myricetin and its main derivatives in myrtle oils. The results pointed out clear differences among extraction methods on myricetin concentration. The oxidative stability of myrtle oils was studied with electron paramagnetic resonance (EPR) spectroscopy highlighting the effect of the extraction method on the oxidation status of the oils and the role of phenolic compounds in the evolution of radical species over time. A principal component analysis applied to LC-MS data highlighted strong differences among phenolic profiles of the oils and highlighted the role of myricetin in the oxidative stability of myrtle oils. Myrtle oil, obtained from the by-products of myrtle liqueur processing industry, extracted with sustainable and green methods might have potential application in food or cosmetic industries. Full article

The genus Capparis is a taxon of difficult delimitation that has several species and ecotypes due to its wide heterogeneity, its extreme phenotypic diversity, and the presence of intermediate forms linked to hybridization phenomena. The Sicilian territory hosts numerous wild and cultivated populations of two spp. Capparis spinosa L. and Capparis orientalis Duhamel, which are ecologically and morphologically distinct. The caper has considerable interest and economic value for its medicinal properties, culinary uses, and cultivation characteristics. It is one of the foods with the highest quercetin content. Quercetin is a flavonol with antioxidant, anti-inflammatory, and immunostimulant properties. Recently, patents and clinical studies have highlighted the inhibitory effect of this compound against several SARS-CoV-2 enzymes (MPro, PLPro, and RdRp). Therefore, the aim of this study was to quantify the amount of quercetin in C. spinosa and C. orientalis by LC-ESI/QTrap/MS/MS and to correlate it with the pedoclimatic features. The results obtained showed that quercetin is more abundant in C. orientalis than in C. spinosa. The highest values of quercetin were recorded in C. orientalis flowers, leaves, and flower buttons of volcanic islands with southwest and east warm exposures. In conclusion, the data acquired can provide a good basis for further scientific investigations to support the identification of possible ecotypes as a source of quercetin for food or pharmaceutical purposes. Full article

The purpose of this research was to assess and utilize the bioactive compounds of garlic nanoparticles (Ga-NPs) as a natural antioxidant in sunflower oil (SFO) stored at 65 ± 1 °C for 24 days. The garlic nanoparticles (Ga-NPs) from the Balady cultivar were prepared, characterized, and added to SFO at three concentrations: 200, 600, and 1000 ppm (w/v), and they were compared with 600 ppm garlic lyophilized powder extract (Ga-LPE), 200 ppm BHT, 200 ppm α-tocopherol, and SFO without Ga-NPs (control). The QTRAP LC/MS/MS profile of Ga-NPs revealed the presence of four organosulfur compounds. Ga-NPs exhibited the highest capacity for phenolic, flavonoid, and antioxidant compounds. In Ga-NP SFO samples, the values of peroxide, p-anisidine, totox, conjugated dienes, and conjugated trienes were significantly lower than the control. The antioxidant indices of SFO samples containing Ga-NPs were higher than the control. The Ga-NPs enhanced the sensory acceptability of SFO treatments up to day 24 of storage. The shelf life of SFO treated with Ga-NPs was substantially increased (presuming a Q₁₀ amount). The results show that Ga-NPs are a powerful antioxidant that improves SFO stability and extends the shelf life (~384 days at 25 °C). Full article

CYP-mediated fast metabolism may lead to poor bioavailability, fast drug clearance and significant drug interaction. Thus, metabolic stability screening in human liver microsomes (HLM) followed by metabolic soft-spot identification (MSSID) is routinely conducted in drug discovery. Liver microsomal incubations of testing compounds with fixed single or multiple incubation time(s) and quantitative and qualitative analysis of metabolites using high-resolution mass spectrometry are routinely employed in MSSID assays. The major objective of this study was to develop and validate a simple, effective, and high-throughput assay for determining metabolic soft-spots of testing compounds in liver microsomes using a single variable incubation time and LC/UV/MS. Model compounds (verapamil, dextromethorphan, buspirone, mirtazapine, saquinavir, midazolam, amodiaquine) were incubated at 3 or 5 µM with HLM for a single variable incubation time between 1 and 60 min based on predetermined metabolic stability data. As a result, disappearances of the parents were around 20–40%, and only one or a few primary metabolites were generated as major metabolite(s) without notable formation of secondary metabolites. The unique metabolite profiles generated from the optimal incubation conditions enabled LC/UV to perform direct quantitative estimation for identifying major metabolites. Consequently, structural characterization by LC/MS focused on one or a few major primary metabolite(s) rather than many metabolites including secondary metabolites. Furthermore, generic data-dependent acquisition methods were utilized to enable Q-TOF and Qtrap to continuously record full MS and MS/MS spectral data of major metabolites for post-acquisition data-mining and interpretation. Results from analyzing metabolic soft-spots of the seven model compounds demonstrated that the novel MSSID assay can substantially simplify metabolic soft-spot identification and is well suited for high-throughput analysis in lead optimization. Full article

A simple sample preprocessing method was developed for the quantitative determination of amantadine (AMT) in human plasma by liquid chromatography-tandem mass spectrometry cubed (LC-MS³). The LC-MS³ system comprised a Shimadzu Exion LC-20AD HPLC pump coupled with a QTRAP 5500 mass spectrometer. First, the plasma samples were pretreated using acetonitrile as the extracting solution to precipitate protein. Next, amantadine and amantadine-d₁₅ (AMT-d₁₅) were separated on an Agilent Poroshell 120 SB-C18 column (4.6 mm × 50 mm, 2.7 μm) using isocratic elution with solvent A (70% 0.1% formic acid) and solvent B (30% acetonitrile) at a flow rate of 0.8 mL/min. The total run time for each sample was 3 min. The system used triple-stage fragmentation transitions at m/z 152.2→135.3→107.4 for AMT quantification in the positive ion mode and m/z 167.0→150.3→118.1 for AMT-d₁₅ quantification. The LC-MS³ assay was linear (r > 0.995) with a concentration range of 50–1500 ng/mL. The lower limit of quantification (LLOQ) was 50 ng/mL, and the intra-day and inter-day accuracies and precisions were less than 8.0% at all concentrations. In addition, the recoveries and matrix effect for AMT in human plasma were within acceptable limits. In terms of stability, AMT had no significant degradation under all conditions. All the results met the requirements of the guidelines of the Food and Drug Administration (FDA) for biological method validation. The novelty of the MS³ assay was that it presented a methodology with higher selectivity and sensitivity. This method was successfully applied to 44 human plasma samples, and the obtained quantitative results were compared with another liquid chromatography-multiple reaction monitoring (LC-MRM) method. The Passing-Bablok regression coefficients and Bland-Altman plot revealed no difference between the LC-MS³ and LC-MRM methods, implying that the developed LC-MS³ method is a reliable and accurate assay for AMT determination in human plasma. These results are also a proof of concept for determining chemicals in biological samples by the LC-MS³ strategy. Full article