Mass Spectrometry Method Research Articles

Validated LC-MS/MS methodology for the quantification of CBD, trace level THCA and UK controlled cannabinoids (Δ9-THC, Δ8-THC, CBN and THCV) in food samples.

Products containing cannabidiol (CBD) have become increasingly popular due to consumer-perceived benefits of improving health and well-being. More specifically in the United Kingdom (UK), CBD food products are categorised as novel foods. For these products to remain on the market, they must have authorisation from the Food Standards Agency on the basis that they are safe, correctly labelled, and do not contain substances classified under controlled drugs legislation in accordance with any existing or future Home Office guidance. This demands for analytical laboratories to be able to accurately measure the CBD concentration using validated methods to confirm correct labelling, as well as the controlled cannabinoid content to ensure the products comply with legislation. To address some of these challenges, this work describes two similar liquid chromatography tandem mass spectrometry (LC-MS/MS) methods which were developed and validated for measuring CBD and trace level controlled cannabinoids in CBD containing foods. The accuracy of the methods developed were tested for the first time through an interlaboratory comparison involving expert laboratories. The methods were applied to a comprehensive study of 148 CBD edible products. In 13 of the products tested (9% of the total) CBD was found below the limit of quantification. Of the remaining 135 products (91% of the total), 66% were found to have detectable amounts of one or more controlled cannabinoids. Of the 13 samples that did not contain detectable levels of CBD, two did contain quantifiable levels of controlled cannabinoids. The validation and sample analysis results reveal intriguing sets of data which help gauge the ongoing narrative surrounding CBD analysis and CBD products available in the UK. The research contributes to the global effort to keep unsafe products off the market.

Impact of Methotrexate and 7-Hydroxymethotrexate Exposure on Renal Toxicity in Pediatric Non-Hodgkin Lymphoma.

7-Hydroxymethotrexate (7-OHMTX) is the main metabolite in plasma following high-dose MTX (HD-MTX), which may result in activity and toxicity of the MTX. Moreover, 7-OHMTX could produce crystalline-like deposits within the renal tubules under acidic conditions or induce renal inflammation, oxidative stress, and cell apoptosis through various signaling pathways, ultimately leading to kidney damage. The objectives of this study were thus to explore the exposure-safety relationship of two compounds and search the most reliable marker for predicting HDMTX nephrotoxicity. A total of 280 plasma concentration data (140 for MTX and 140 for 7-OHMTX) for 60 pediatric patients with non-Hodgkin lymphoma (NHL) were prospectively collected. Plasma MTX and 7-OHMTX concentrations were determined using a high-performance liquid chromatography tandem mass spectrometry (HPLC-MS/MS) method. A nonlinear mixed effect model approach was used to build a joint population pharmacokinetic (PopPK) model. After validation, the model estimated the peak concentration (Cmax) and area under the curve within the initial 48 h (AUC0-48h) of the patients after drug administration by Bayesian feedback. The receiver operating characteristic (ROC) curves were generated to identify an exposure threshold associated with nephrotoxicity. A three-compartment chain model (central and peripheral compartments for MTX and central compartment 7-OHMTX) with the first-order elimination adequately characterized the invivo process of MTX and 7-OHMTX. The covariate analysis identified that the aspartate aminotransferase (AST) was strongly associated with the peripheral volume of distribution of MTX. Moreover, the Cmax of MTX and 7-OHMTX showed significant differences (p < 0.0001, p = 0.0472, respectively) among patients with or without nephrotoxicity. Similarly, individuals with nephrotoxicity also exhibited substantially higher ratio of 7-OHMTX to MTX peak concentration and the sum of MTX + 2.25 times the concentration of 7-OHMTX (p < 0.0001, p = 0.0426, respectively). By ROC analysis, the Cmax of MTX and 7-OHMTX had the greatest area under the curve (AUC) values (0.769 and 0.771, respectively). A Cmax threshold of 9.26 μmol/L for MTX or a Cmax threshold of 0.66 μmol/L for 7-OHMTX was associated with the best sensitivity/specificity for toxicity events (MTX: sensitivity = 0.886; specificity = 0.70; 7-OHMTX: sensitivity = 0.886; specificity = 0.70). We demonstrated that the Cmax of MTX and 7-OHMTX were the most reliable markers associated with nephrotoxicity and proposed a Cmax threshold of 9.26 μmol/L for MTX and 0.66 μmol/L for 7-OHMTX as the point with a high risk of nephrotoxicity. Altogether, this study may contribute to crucial insights for ensuring the safe administration of drugs in pediatric clinical practice.

Determination of eight phthalate metabolites in urine of pregnant women and evaluation of neonatal birth outcomes based on solid-phase extraction-high performance liquid chromatography-tandem mass spectrometry

Phthalates (PAEs) are endocrine-disrupting chemicals that are widely present in everyday life and enter the human body through various pathways. The release of PAEs into the environment through pathways that include leaching, evaporation, abrasion, and the use of personal care products exposes humans to PAEs via ingestion, inhalation, and dermal absorption. Pregnant women, as a particularly vulnerable population, risk adverse newborn growth and development when exposed to PAEs. While the concentrations of PAEs in urine reflect recent exposure levels in humans, urinary levels of phthalate metabolites (mPAEs) are commonly used as biomarkers of internal exposure owing to the relatively short biological half-lives of PAEs (<24 h). In this study, we developed a solid-phase extraction-high performance liquid chromatography-tandem mass spectrometry (SPE-HPLC-MS/MS) method for simultaneously detecting eight mPAEs in the urine of pregnant women. Urine samples were enzymatically hydrolyzed with β-glucosidase and then purified using the Bond Elut Plexa SPE column, with subsequent elution, concentration, and redissolved performed prior to HPLC-MS/MS. Separation was achieved using an Agilent Eclipse Plus C18 column (100 mm×3 mm, 3.5 μm), with gradient elution performed using 0.1% acetic acid aqueous solution and 0.1% acetic acid acetonitrile as mobile phases. Multiple reaction monitoring (MRM) mode was used for detection, with quantification performed using the internal-standard method. Good linearities were obtained in the range of 0.1-200 ng/mL for the eight mPAEs, with limits of detection (LODs) and quantification (LOQs) of 0.015-0.048 and 0.050-0.160 ng/mL, respectively. The eight mPAEs exhibited recoveries of 80.2%-99.7% at three spiked levels (1, 10, and 50 ng/mL). This method was subsequently used to analyze the eight mPAEs levels in urine samples of 497 pregnant women from the Ezhou Maternity and Child Health Care Hospital. The participants exhibited widespread exposure to PAEs, with monobutyl phthalate (MBP) showing the highest median level of 104.46 ng/mL, and monobenzyl phthalate (MBzP) showing the lowest (0.22 ng/mL). In addition, this study assessed neonatal birth outcomes. Linear regression modeling revealed that gestational age decreased by 0.11 weeks (95% confidence interval (CI): -0.18--0.03) for every natural-log (ln) increase in the level of monoethyl phthalate (MEP) in urine samples of pregnant woman. Moreover, the birth weight decreased by 39.28 g (95% CI: -76.48--2.09) and 39.62 g (95% CI: -73.73--5.52), for every ln increase in mono(2-ethyl-5-oxohexyl) phthalate (MEOHP) and mono(2-ethylhexyl) phthalate (MECPP) levels, respectively. The developed method is characterized by its simplicity, low LODs, high accuracy, and precision. This study provides clear evidence that PAE exposure during pregnancy negatively affects newborn growth and development by measuring the levels of eight mPAEs in the urine of pregnant women and linking these findings to neonatal outcomes. Further large-scale cohort studies are needed to validate these findings, along with mechanistic studies using animal models or in-vitro systems that elucidate the biological pathways through which mPAEs contribute to adverse birth outcomes.

Development and validation of a UHPLC-MS/MS method for the quantitative analysis of trans-ISRIB in human plasma.

The integrated stress response (ISR) is a cellular defense mechanism activated under stress conditions. When the ISR is activated, it slows the production of proteins, the building blocks that cells need to function. Trans-integrated stress response inhibitor (trans-ISRIB) is a compound that can reverse the effects of ISR activation, showing promise for treating neurodegenerative diseases. The preclinical and clinical evaluation of trans-ISRIB necessitates a reliable analytical method. This study presents the development and validation of an ultra-high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) method for the quantitative analysis of trans-ISRIB in human plasma, conforming to the U.S. FDA's guidelines for bioanalytical method validation. The method developed utilizes a liquid-liquid extraction procedure to prepare plasma samples with a spiked internal standard (IS). The extracts containing trans-ISRIB and the IS were dried under nitrogen, reconstituted in the mobile phase, and separated on a Waters XSelect HSS T3 column under isocratic conditions with a mobile phase containing 0.1% acetic acid in 70% methanol aqueous solution at a flow rate of 0.500mL/min. Detection and quantification were accomplished using a positive electrospray ionization tandem mass spectrometer (ESI+-MS/MS) operated in multiple-reaction-monitoring (MRM) mode. The method demonstrated a linear calibration range for trans-ISRIB concentrations from 0.500 to 1.00x103 nM, with high specificity, precision, accuracy, and recovery. This method addresses a significant analytical gap, offering a robust tool for quantifying trans-ISRIB in human plasma. Chemical compounds studied in this article: 2-(4-chlorophenoxy)-N-[4-[[2-(4-chlorophenoxy)acetyl]amino]cyclohexyl]acetamide (trans-ISRIB) (CAS # 1597403-47-8); 2-(4-chlorophenoxy)-N-(2-{[(4-chlorophenoxy)acetyl]amino}ethyl)acetamide (CAS # 327071-30-7).

Quantification and clinical performance of serum parathyroid hormone 1-84 via immunocapture coupled to LC-MS/MS in chronic renal failure.

Accurate measurement of serum parathyroid hormone (PTH) is crucial for diagnosing and managing endocrine and osteological diseases. Conventional immunoassay methods struggle with cross reactivity issues between full-length PTH and truncated fragments or post-translationally modified forms. Both the standardization of PTH assays and the peptide's stability are concerning. This study addresses these issues by establishing an immunocapture coupled with liquid chromatography-tandem mass spectrometry (LC-MS/MS) method to precisely quantify PTH1-84. PTH1-84 was isolated from one mL serum samples by immunocapture on a polystyrene bead and eluted from matrix, then quantitated by LC-MS/MS. The results from 268 serum samples were compared to an intact PTH immunoassay. The assay's linear range was 5.0-1000.0 pg/mL. The intra-assay coefficients of variation (CVs) ranged from 3.2 % to 6.8 %, and the inter-assay CVs ranged from 4.6 % to 9.5 %. The extraction efficiencies were 98.0 %-100.5 %, with no significant matrix effects observed after internal standard correction. The correlation coefficient between LC-MS/MS and immunoassay was 0.989, but the bias between the methods was substantial. Nevertheless, the immunocapture purification coupled LC-MS/MS method offers a promising approach for accurate PTH measurement.

Nivolumab plasma concentration and clearance associated with overall survival in patients with renal cell carcinoma

BackgroundNivolumab is an immune checkpoint inhibitor (ICI) that selectively inhibits programmed cell death protein 1 activation, restoring antitumor immunity. ICIs are indicated for various types of advanced solid tumors; however,...

Identification of 3-methoxytyramine as a specific biomarker for beet-sugar-fed honey: A two year surveillance study in South Korea

Honey is highly vulnerable to food fraud, and there are growing concerns about product authenticity. The commonly used stable carbon isotope ratios in the Calvin (C3) and Hatch–Slack (C4) photosynthesis cycles in plant feed cannot distinguish between beet-sugar-fed honey and natural honey. However, 3-methoxytyramine (3-MT) can be used as specific biomarker for identifying adulteration of beet-sugar-fed honey. In this study, we developed an isotope dilution-liquid chromatography/tandem mass spectrometry method to quantify 3-methoxytyramine (3-MT) in beet-sugar-fed honey. Solid phase extraction was used to quantify 3-MT and to reduce the matrix interference from honey samples. The limit of quantification was 1 μg/kg. The accuracy (recovery, %) and precision (relative standard deviation) values, determined in three laboratories, were 100–114 % and 1.25–9.07, respectively. Moreover, 3-MT was detected (1.07–10.7 μg/kg) in 144 out of 3,172 samples collected from different parts of Republic of Korea over 2021 and 2022. The mean 3-MT concentration of seven honey samples fed with 100 % beet sugar was 5.32 μg/kg. The results of our study validated the accuracy and sensitivity of the proposed analytical method was established for quantification of 3-MT in honey samples. Additionally, our study demonstrated that the 3-MT as a reliable biomarker for beet-sugar-fed honey adulteration. Thus, our study presents a novel mass spectrometric approach for authentic honey discrimination with potential application in honey authenticity testing.

Rapid 235U/238U determination by matrix assisted ionization–time-of-flight mass spectrometry

Matrix assisted ionization is an effective tool for producing rapid and accurate uranium isotope ratios with minimal laboratory infrastructure needs compared to traditional mass spectrometry methods.

A Novel Liquid Chromatography-Tandem Mass Spectrometry Method to Quantify Tryptophan and Its Major Metabolites in Serum to Support Biomarker Studies in Patients with Cancer Undergoing Immunotherapy.

Tryptophan (TRP) is an essential amino acid crucial for the production of many bioactive compounds. Disturbances in TRP metabolism have been revealed in various diseases, many of which are closely related to the immune system. In recent years, we have focused on finding blood-based biomarkers of successful immunotherapy in cancer. Thus, we aimed to develop a robust liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for TRP and its metabolites that could be used in biomarker studies. Although analyzing TRP derivatives in biological matrices is not a new topic, we encountered multiple challenges during method development. One of them was the phenomenon of cross-interferences between the analyzed molecules, which has not been explored in most published papers. We noticed that injecting a pure single-compound solution often generated a signal in the other compounds' MS/MS channels. Specifically, TRP generated unexpected peaks in the channel for kynurenine, kynurenic acid, and xanthurenic acid, while kynurenine generated peaks in the channel for kynurenic acid. We also recorded a mutual cross-talk between kynurenine and isotope-labeled TRP. Different origins of the observed cross-signal contribution were proposed. This paper draws attention to investigating cross-interferences in LC-MS/MS, especially when structurally related compounds will be analyzed. Despite all the challenges, the method was successfully validated according to international guidelines (EMA/ICH), and its applicability was confirmed in a pilot study including 20 patients with lung cancer undergoing chemoimmunotherapy.

Cocaine-Induced Remodeling of the Rat Brain Peptidome: Quantitative Mass Spectrometry Reveals Anatomically Specific Patterns of Cocaine-Regulated Peptide Changes.

Addiction to psychostimulants, including cocaine, causes widespread morbidity and mortality and is a major threat to global public health. Currently, no pharmacotherapies can successfully treat psychostimulant addiction. The neuroactive effects of cocaine and other psychostimulants have been studied extensively with respect to their modulation of monoamine systems (particularly dopamine); effects on neuropeptide systems have received less attention. Here, we employed mass spectrometry (MS) methods to characterize cocaine-induced peptidomic changes in the rat brain. Label-free peptidomic analysis using liquid chromatography coupled with tandem MS (LC-MS/MS) was used to describe the dynamic changes of endogenous peptides in five brain regions (nucleus accumbens, dorsal striatum, prefrontal cortex, amygdala, and hypothalamus) following an acute systemic cocaine challenge. The improved sensitivity and specificity of this method, coupled with quantitative assessment, enabled the identification of 1376 peptides derived from 89 protein precursors. Our data reveal marked, region-specific changes in peptide levels in the brain induced by acute cocaine exposure, with peptides in the cholecystokinin and melanin-concentrating hormone families being significantly affected. These findings offer new insights into the region-specific effects of cocaine and could pave the way for developing new therapies to treat substance use disorders and related psychiatric conditions.

Hydrothermal uranium mineralization in the Tuyukan ore field (Northern Transbaikal region): time of formation of pitchblende mineralization and age of the source of the matter

Based on the study of local volumes of minerals using classical methods of isotope dilution and thermal ionization mass spectrometry (U–Pb ID TIMS), an isotopic study of pitchblende and galena was carried out at the Natali uranium ore occurrence (Northern Transbaikalia). For the first time, it was shown that pitchblende mineralization at the ore occurrence is Late Silurian (422±4 million years) age. Its formation is associated with early-mid Paleozoic collisional tectogenesis in the area of the Tonod uplift, accompanied by increased fluid activity. The results of analyzing the Pb isotopic composition in galena associated with pitchblende showed that it is enriched in the radiogenic isotopes 206Pb and 207Pb, the source of which was ancient (Precambrian) concentrations of uranium.

Effects of vaginal estrogen on serum estradiol during aromatase inhibitor therapy in breast cancer patients with vulvovaginal atrophy: a prospective trial.

This study aimed to analyze changes in serum estradiol (E2) levels during concurrent vaginal estradiol therapy and adjuvant letrozole in postmenopausal breast cancer (BC) patients with vulvovaginal atrophy (VVA). Secondary objectives included assessing the effects of therapy on vaginal atrophy, quality of life (QoL) and menopause-related symptoms. 20 postmenopausal patients undergoing adjuvant letrozole therapy and experiencing VVA symptoms were treated with vaginal estradiol for 12weeks. Gynecologic examination and symptom screening were conducted at baseline and after 12weeks. Serum E2 levels were analyzed at baseline, and at two, four, eight, and 12weeks. E2 levels were measured using both a routine liquid chromatography-tandem mass spectrometry (LC-MS/MS) method and a highly sensitive (hsE2-MS) LC-MS/MS method. At baseline, serum E2 levels, measured with hsE2-MS, were below the lower limit of quantification (LLOQ) in all patients. E2 remained below LLOQ throughout the treatment period in three patients (15%). Persistent E2 elevation above LLOQ was observed in six patients (30%), while isolated E2 elevations occurred in 10 patients (50%). One patient experienced transient E2 elevation in two sporadic measurements. Serum E2 variations were shown by using both LC-MS/MS methods. Vaginal pH, vaginal maturation index (VMI), and VVA symptoms significantly improved during treatment. Intravaginal estradiol therapy (10ug) during adjuvant letrozole resulted in transient increases in systemic E2 levels among early BC patients with VVA. Highly sensitive LC-MS/MS is a promising method for monitoring E2 levels during aromatase inhibitor (AI) therapy.

Lysosphingolipid Quantitation in Plasma and Dried-Blood Spots Using Targeted High-Resolution Mass Spectrometry.

Sphingolipidoses are rare inherited metabolic diseases belonging to lysosomal diseases. Early and accurate diagnosis is crucial for effective management and treatment. In this study, we aimed to develop a robust method to accelerate the diagnosis of these sphingolipidoses using dried blood spots and plasma. We employed high-resolution mass spectrometry coupled with liquid chromatography (LC-HRMS) to analyze 6 lysosphingolipids (GlcSph/Psychosine, LysoGb3, LysoSM, LysoSM509, LysoGM1, and LysoGM2) on dried blood spots and plasma samples. The method was used to measure the lysosphingolipid levels in a group of 30 control subjects and 204 samples from patients with sphingolipidoses (61 dB and 143 plasma) including Fabry, Gaucher, GM2 Gangliodosis, Niemann-Pick type A/B, and Niemann-Pick type C. The developed multiplex LC-HRMS method demonstrated linearity, precision, and quantification performances particularly for GlcSph/Psychosine and LysoGb3 on samples including controls and patients with sphingolipidoses. LysoSM showed recovery variability, wherease LysoGM1 and LysoGM2 showed higher matrix effect. Our study presents a high-resolution mass spectrometry method along with the established cutoff values, providing a valuable tool for targeted screening, accurate diagnosis, and monitoring sphingolipidoses. Furthermore, DBS showed reliable results that lay the path to a broader adoption for screening these diseases.

Liquid Chromatography‐Tandem Mass Spectrometry Analysis and Pharmacokinetics of Doxorubicin and Its Prodrug in Rats After Administration of Nanoparticles

ABSTRACTTo further improve the dilemma facing the clinical application of doxorubicin (DOX), we designed a cathepsin B sensitive DOX prodrug albumin nanoparticle (NP). To evaluate the pharmacokinetics of prodrug albumin NPs (C16‐GFLG‐PAB‐DOX NPs), we developed and validated a liquid chromatography‐tandem mass spectrometry method for the same time analysis of DOX and its fatty acid prodrug (C16‐GFLG‐PAB‐DOX) in rat plasma. The method uses the precipitated protein method to extract analytes from rat plasma. The validated method on selectivity, linearity (r ≥ 0.992), precision (relative standard deviation: 3.5%–9.5%), accuracy (relative error: −2.9% to 3.5%), extraction recovery (80.2% to 90.4%), matrix effect (87.8% to 96.6%), and stability in different conditions were satisfied. Therefore, the pharmacokinetic behavior of C16‐GFLG‐PAB‐DOX NPs in rats was investigated using this analytical approach. The outcome of the pharmacokinetic study illustrated that prodrug albumin NPs are promising to alleviate the problems associated with DOX in clinical applications.

Quantitative Determination of a Series of Oxysterols by an Optimized LC-MS/MS Analysis in Different Tissue Types.

Oxysterols, as metabolites of cholesterol, play a key role in cholesterol homeostasis, autophagosome formation, and regulation of immune responses. Disorders in oxysterol metabolism are closely related to the pathogenesis of neurodegenerative diseases. To systematically investigate the profound molecular regulatory mechanisms of neurodegenerative diseases, it is necessary to quantify oxysterols and their metabolites in central and peripheral biospecimens simultaneously and accurately. However, there are a lot of unsolved problems with the existing methods, such as the hindrance of applying a single method to different biological specimens or the challenge of simultaneous quantification due to differential groups on the ends of the oxysterol side chains. Herein, according to the physicochemical properties and structure of oxysterols, an optimized liquid chromatography-tandem mass spectrometry method for the quantification of oxysterols was established by optimizing the sample preparation process, chromatographic conditions, mobile phase pH, and solvent selection. Seven oxysterols were detected by this method, including 27-hydroxycholesterol, 7α-hydroxycholesterol, 7α,27-dihydroxycholesterol, 7-dehydrocholesterol, 7α-hydroxy-3-oxo-4-cholestenoic acid, 3-hydroxy-5-cholestenoic acid, and 24(S)-hydroxycholesterol. Non-derivatization extraction with methyl tert-butyl ether was used for different biospecimens, followed by simultaneous chromatographic separation of oxysterols on a phenyl hexyl column. By repeated validation, this method exhibited satisfactory linearity, precision, recovery, sensitivity, repeatability, and stability, and it was successfully applied to the detection of oxysterols in the plasma, cerebral cortex, and liver of mouse. In summary, our optimized method enables concurrent analysis and quantification of oxysterols and their metabolites in various biospecimens, presenting a broad range of applicability.

Development and Validation of a Robust and Straightforward LC-MS Method for Measuring Taurine in Whole Blood and Plasma of Dogs and Reference Intervals Calculation

Several studies have highlighted the essential role of taurine in maintaining the health of small animals, particularly dogs. Taurine deficiency has been linked to various health issues, especially in certain dog breeds. Therefore, accurately assessing taurine levels in canine blood is crucial for diagnosing and monitoring these conditions. In this study, we present the development of a novel liquid chromatography–tandem mass spectrometry (LC-MS/MS) method for rapidly quantifying taurine concentrations in dog whole blood and plasma. The method was validated according to current guidelines, showing excellent accuracy, precision, and sensitivity across a wide concentration range. Specifically, the limit of quantification was set at 80 nmol/mL for whole blood and 8 nmol/mL for plasma, ensuring the method’s reliability for both matrices. The application of this validated technique to blood samples of healthy dogs allowed for the establishment of reference intervals for taurine concentrations (148 to 495 nmol/mL for whole blood; 42 to 183 nmol/mL for plasma). Due to its robustness and simplicity, this method represents a valuable tool, supporting its routine use in health assessments and enabling more effective monitoring of taurine status in dogs.

Microbiota of complete removable dentures

BACKGROUND: Complete dentures are a valid treatment option in edentulous patients. However, the average service life of complete dentures is 4–5 years, which is frequently due to microbial contamination. AIM: To perform quantitative and qualitative assessment of microbiota on the surface of complete dentures after 1 and 4 years of use. MATERIALS AND METHODS: The study included 40 fully edentulous patients (К08.1) who used acrylic complete dentures for no more than 5 years. There were two groups (n=20 each) based on the duration of use of complete dentures: 1 year in Group 1 and 4 years in Group 2. The microbiota composition was examined by mass spectrometry. The Statistiсa 13 package was used for statistical processing of the study findings. For multiple comparisons, the parametric t-test with Bonferroni correction was used to assess intergroup differences, with p=0.05 as the critical significance level. RESULTS: Microbial contamination increased in all examined patients after using complete dentures for 1 to 4 years. Cocci and bacilli counts increased from 56±5 (105 cells/g) (р=0.03) to 107±8 (105 cells/g) (р=0.04). Anaerobe counts increased from 68±6 (105 cells/g) (р=0.0002) to 102±9 (105 cells/g) (р=0.0002). Actinobacteria counts increased from 30±3 (105 cells/g) to 143±12 (105 cells/g) (р=0.003). Gram-negative rod counts increased from 4±1 (105 cells/g) (р=0.0005) to 24±2 (105 cells/g) (р=0.0006). Yeast and mold counts increased from 977±90 (105 cells/g) (р=0.0003) to 1,587±136 (105 cells/g) (р=0.003). CONCLUSION: Within 4 years, yeast and mold counts increased by 91%, actinobacteria counts by 61%, gram-negative rod counts by 500%, and anaerobe counts by 50% in all patients. The study findings indicate that microbial contamination of dentures is directly related to the duration of their use.

Monitoring Fusarium toxins from barley to malt: Targeted inoculation with Fusarium culmorum.

Molds of the genus Fusarium infect nearly all types of grain, causing significant yield and quality losses. Many species of this genus produce mycotoxins, which pose significant risks to human and animal health. In beer production, the complex interaction between primary fungal metabolites and secondarily modified mycotoxins in barley, malt, and beer complicates the situation, highlighting the need for effective analytical methods to quickly and accurately monitor these toxins. We developed and validated a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method to simultaneously analyze 14 Fusarium toxins, including modified forms (deoxynivalenol (DON), DON-3-glucoside, 3-acetyl-DON, 15-acetyl-DON, nivalenol, fusarenone X, HT-2 toxin, T-2 toxin, the enniatins A, A1, B, B1, beauvericin, and zearalenone) in barley and throughout the malting process. Stable isotope dilution assays (SIDAs) and matrix-matched calibration were used for quantification. A micro-malting setup was established to produce Fusarium-contaminated barley malt under reproducible conditions using targeted inoculation with F. culmorum. Mycotoxins were quantified throughout the malting process and compared to the content of fungal DNA. Further, the impact of various malting parameters was investigated, thus revealing that different malting scenarios exhibited different toxin enrichment patterns. We demonstrated that mycotoxin concentration and the ratio of DON to DON-3-glucoside changed throughout the malting processes, depending on fungal spore concentrations, germination temperature, and malting temperature. The study highlights the complexity of mycotoxin dynamics in malt production and the importance of optimized processing conditions to minimize toxin levels in final malt products.

Quantification of multi-pathway metabolites related to folate metabolism and application in natural population with MTHFR C677T polymorphism.

Folate, serving as a crucial micronutrient, plays an important role in promoting human growth and supporting transformations to a variety of metabolic pathways including one-carbon, pyrimidine, purine, and homocysteine metabolism. The 5,10-methylenetetrahydrofolate reductase (MTHFR) enzyme is pivotal in the folate metabolic pathway. Polymorphism in the MTHFR gene, especially C677T, was associated with decreased enzyme activity and disturbance of folate metabolism, which is linked to various diseases including birth defects in newborns and neural tube abnormalities. However, the detailed metabolic disturbance induced by MTHFR C677T polymorphism is still elusive. In this study, a novel liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was developed for the precise quantification of 93 metabolites from six important metabolic pathways related to folate metabolism. The method characteristics demonstrated high accuracy and precision, with r2 values ranging from 0.981 to 1.000 for all metabolites. Then the impact of the MTHFR C677T polymorphism on folate metabolism was further investigated, revealing a significant reduction in the level of 5-methyltetrahydrofolate and abnormal levels of metabolites associated with DNA synthesis pathways in individuals carrying the mutation. These data highlight the pivotal role of folic acid supplementation for individuals with the MTHFR C677T polymorphism to mitigate health risks and show the value of precision measurement of folate-related metabolites.

Ochratoxin A and AFM1 in Cheese and Cheese Substitutes: LC-MS/MS Method Validation, Natural Occurrence, and Risk Assessment.

Cheese is vulnerable to contamination with mycotoxins, particularly ochratoxin A (OTA) and aflatoxin M1 (AFM1). This study aims to develop and validate an analytical method for the detection and quantification of OTA and AFM1 in cheese and to assess their prevalence and associated risks. A liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was validated for detecting these mycotoxins in 41 cheese samples, including firm-ripened, spreadable, and plant-based alternatives. The results showed that OTA was detected exclusively in grated Parmigiano Reggiano cheese, while AFM1 was found in both Parmigiano Reggiano and Pecorino cheeses. This study goes beyond analytical method development by providing a preliminary exposure assessment and risk characterization for OTA and AFM1 in cheese, bridging the gap between analytical chemistry and public health implications. This study identified potential health risks associated with OTA, particularly for children and adolescents categorized as high consumers of Parmigiano Reggiano cheese. The findings underscore the need for monitoring of OTA and AFM1 in cheese and further research to establish regulatory limits for these contaminants.