LC-tandem MS Research Articles

Proteomics Approach to Identify Serum Biomarkers Associated with Gastric Cancer in South Indian Tamils.

Identifying cancer-specific biomarkers is a crucial step in the disease screening process at a very early stage of tumor development. In recent years, Quantitative proteomic approaches have gained importance in identifying novel candidate markers in cancer. Gastric cancer has always been known as a life-threatening medical condition with high mortality rates. The objective of our research is to adapt serum samples from Indian gastric cancer patients to identify and understand the differentially regulated proteins in comparison with healthy individuals. A total of 30 serum isolates from gastric cancer patients and healthy individuals were obtained and subjected to 2-D Gel electrophoresis, and Tandem LC-MS analysis revealed 12 differentially expressed protein spots. The functional properties of identified proteins were further analyzed using PANTHER and STRING databases. The differentially expressed protein spots were identified as three candidate proteins: Haptoglobin, Prohibitin, and Apolipoprotein. The protein interaction studies reveal that the haptoglobin fragments were upregulated, and the remaining two prohibitin and Apolipoprotein were down-regulated in gastric cancer patients. All the proteins identified as biomarkers were found to be involved in regulating cell proliferation and stabilization of oxidative metabolism in the liver; therefore, differential regulation plays a crucial role in gastric cancer progression.

Red meat intake, faecal microbiome, serum trimethylamine N-oxide and hepatic steatosis among Chinese adults.

Emerging evidence suggests a detrimental impact of high red meat intake on hepatic steatosis. We investigated the potential interplay between red meat intake and gut microbiome on circulating levels of trimethylamine N-oxide (TMAO) and hepatic steatosis risk. This cross-sectional study was conducted in a representative sample of 754 community-dwelling adults in Huoshan, China. Diet was collected using 4 quarterly 3 consecutive 24-h dietary (12-day) recalls. We profiled faecal microbiome using 16S ribosomal RNA sequencing and quantified serum TMAO and its precursors using LC-tandem MS (n = 333). We detected hepatic steatosis by FibroScan. The adjusted odds ratios (aORs) and 95% confidence intervals (CIs) were calculated using logistic regression. TMAO levels but not its precursors were positively associated with the likelihood of hepatic steatosis (aOR per 1-SD increment 1.86, 95% CI 1.04-3.32). We identified 14 bacterial genera whose abundance was associated with TMAO concentration (pFDR < .05) belonging to the phyla Firmicutes, Bacteroidetes, Actinobacteria and Proteobacteria families. Per 10 g/day increase in red meat intake was positively associated with TMAO levels among participants who had higher red meat intake (>70 g/day) and higher TMAO-predicting microbial scores (TMS, β = .045, p = .034), but not among others (pinteraction = .030). TMS significantly modified the positive association between red meat and steatosis (pinteraction = .032), with a stronger association being observed among participants with higher TMS (aOR 1.30, 95% CI 1.07-1.57). The bacterial genera that predicted TMAO levels may jointly modify the association between red meat intake and TMAO levels and the subsequent risk of hepatic steatosis.

Assessment of organic pollutants in petroleum refinery wastewater by LC-MS analyzer

The release of toxic and hazardous effluents from petroleum refining companies seriously threatens the poisoning of water bodies. Thus, it is necessary to determine an accurate treatment process by understanding the nature of wastewater. This work uses Liquid Chromatography-Mass Spectroscopy (LCMS), including LC-Tandem MS (LCMS/MS), ion trap (IT), and quadrupole-time-of-flight (Q-TOF) to comprehend the composition of petroleum refinery effluent. Many polar metabolites available, even as a trace fraction, can be found using advanced LCMS with defined molecular structure, which may or may not be itemized in CAS. Based on the number of carbon atoms (C1-C48), chemical reactivity (saturated, unsaturated, and aromatics), and functional group (hydrocarbons containing oxygen only and other minerals along with oxygen), substances are identified. Using LCMS/MS, 170 organic components, 77.10% negative and 22.90% positive metabolites, are quantified. A range of carbon between C8 and C48 is identified as having 6.50% C8-C12, 30% C13-C17, and 63.50% C18-C48. Higher molecular mass hydrocarbons from C22-C48 are less soluble in water and are more positively polarized, while C8-C21 hydrocarbons are more soluble under negative polarity. The principal ingredient is documented as consisting primarily of aromatic chemicals (72.30%), the majority of which are found as harmful metabolites. Further, 83% of the hydrocarbons are acknowledged to contain solely oxygen compared to other minerals in petroleum effluents. Overall, it was concluded that the direct discharge of petroleum refinery effluents without treatment may cause various health and environmental issues.

Associations of dietary advanced glycation end products with liver steatosis via vibration controlled transient elastography in the United States: a nationwide cross-sectional study.

Large population-based studies for the associations between dietary advanced glycation end products (dAGEs) intake and liver steatosis remain lacking. It is necessary to clarify the relationship of dAGEsintake with liver steatosis through the National Health and Nutrition Survey (NHANES). A total of 5856 participants in the NHANES 2017-2018 were included. The dietary AGEs intake, including ε-(carboxymethyl)lysine(CML), Nε-(1-carboxyethyl)lysine (CEL), and Nδ-(5-hydro-5-methyl-4-imidazolon-2-yl)-ornithine (MG-H1) were estimated using the combination of ultra-performance LC-tandem MS dietary AGEs database and two 24-h dietary recall interviews. Liver steatosis was assessed by controlled attenuation parameter via transient elastography. Logistic regression model was adopted to explore the relationships between dAGEs intake and hepatic steatosis. Compared with individuals of total dAGEs, CML, MG-H1 in the lowest tertile, those in the highest tertile had highest risk of hepatic steatosis, and the corresponding odds radios(ORs) (95% confidence interval(CI)) were 1.37 (1.01, 1.84), 1.36 (1.04,1.78) and 1.40 (1.06, 1.85), respectively. Subgroups analysis found that the positive association between dAGEs, CML, CEL and MG-H1 and hepatic steatosis appeared stronger in subjects with obesity and those with abnormal waist circumference (WC). There was a positive correlation between dAGEs, CML, MG-H1, and hepatic steatosis, and this association mainly existed in subjects with obesity and those with abnormal WC. Dietary AGEs restriction might be of high priority for subjects with obesity for the prevention of fatty liver disease. Further longitudinal studies are required to confirm the causal associations and explore the potential mechanisms.

Higher dietary advanced glycation products intake is associated with increased risk of dementia, independent from genetic predisposition

Dietary advanced glycation end products (AGEs) might exert adverse effects on cognition. The associations between dietary AGEs and long-term risk of dementia are yet to be assessed in large population studies. We aimed to explore whether elevated dietary AGEs intake is associated with increased risk of dementia, and whether this association might be affected by genetic risk. A prospective cohort study, which included a total of 93,830 participants (aged≥ 50 years) free from dementia at baseline of the UK Biobank study (2006-2010) and had at least two 24-h dietary assessments and were followed up until 2021. Dietary AGEs, including Nε-(1-Carboxyethyl)-l-lysine (CEL), Nε-(carboxymethyl) lysine (CML), and Nδ-(5-hydro-5-methyl-4-imidazolon-2-yl)-ornithine (MG-H1) were estimated via averaged data from the multiple 24-h food assessments according to the ultra-performance LC-tandem MS based dAGEs database. Incidence of all-cause dementia was ascertained through hospital inpatient and mortality records. Multivariable Cox regression models were utilized to estimate hazards ratios (HRs) and 95% confidence interval (CI) of dementia risk associated with dietary AGEs. During a median follow-up of 11.9 years, 728 participants developed dementia. In multivariable adjusted model, when comparing the highest with the lowest tertile of intake level, HRs (95% CI) of dementia were 1.43 (1.16, 1.76) for total AGEs Z score, 1.53 (1.25, 1.89) for CEL, 1.27 (1.03, 1.56) for CML and 1.24 (1.02, 1.52) for MG-H1 (all P trend<0.01). There was no significant interaction between dietary AGEs intake, genetic risk and APOE ε4 carrier status for dementia. Higher intakes of dietary AGEs including CEL, CML and MG-H1 were associated with a higher risk of dementia, independent from genetic risk, highlighting the significance of dietary AGEs restriction for dementia prevention.

Off-line two-dimensional LC–tandem MS of menaquinones from thermophilic bacteria

Thermophilic bacteria of four genera in contrast to the commonly used production strains such as Bacillus subtilis, produce homologs other than menaquinone (MK) with seven isoprene units. The number of isoprene units and the configuration of double bonds are essential factors for their biological activity. The goal was to obtain a strain of bacteria that produces a wide range of MK homologs and only all-trans geometrical isomers, which was the strain G. kaustophilus. Using off-line two-dimensional LC–tandem MS in columns with the RP18 phase and the COSMOSIL cholester phase (separation according to the geometric configuration of double bonds) it was shown that thermophilic bacteria grown at different temperatures produce only all-trans isomers of menaquinones from MK-5 (menaquinone with five isoprenyl units) to MK-15 (fifteen isoprenyl units). Therefore, G. kaustophilus appears to be a biotechnologically important strain produces only trans isomers and additionally homologs from 5 to 15 isoprene units.

Prevalence and Detection of Pharmaceuticals in Hospital Wastewater: A Case of Referral and District Hospitals in Zanzibar

Despite the great efforts taken by the developed and some developing countries in managing the release of pharmaceuticals from their primary sources to the environment; the pharmaceutical wastewater management in Zanzibar-Tanzania still has not received much attention as a bulk of pharmaceuticals and their metabolites are released to the environment without proper treatment. Therefore, this study has scrutinized the incidence of pharmaceuticals in wastewater released from the referral and district hospitals in Zanzibar, to ascertain the levels of pharmaceuticals present in hospital wastewater. Purposive sampling was implemented to collect a total of seventy-two (72) wastewater samples in a period of six months (March-August 2022). Samples were collected in the effluent of wastewater-streams (Mnazi Mmoja Hospital, MMH) and pit latrines (Kivunge District Hospital (KDH) and Makunduchi District Hospitals (MDH)). The pharmaceuticals in these samples were obtained via the solid phase extraction after which they were analyzed using the LC-tandem MS (Agilent 1,290 LC coupled to 6,460-triple quadrupole MS). The limit of detections (LODs) and limit of quantitations (LOQs) for the determination of pharmaceuticals were in the range of 0.021-0.037 μg L-1 and 0.033-0.059 μg L-1, respectively. The detected analytes belonged to antibiotics, anti-inflammatory, benzodiazepine, antipsychotic, antipyretic and anticonvulsant (anti-epileptic) drugs. Diclofenac (DIC), paracetamol (PCT), ciprofloxacin (CIP), sulfamethoxazole (SMZ) and azithromycin (AZM) were detected at higher concentrations (&gt; 0.25 μg L-1) in wastewater samples collected from KDH and MDH. Lower concentrations of pharmaceuticals (&lt; 0.10 µg L-1) were identified in MMH wastewater samples. These present findings provide estimable information on the incidence of pharmaceuticals in wastewater that can assist in strengthening the environmental strategies for the protection of marine and terrestrial life from pharmaceutical pollution in Zanzibar.

A fatal mono-intoxication with 4-fluoroisobutyrylfentanyl: Case report with postmortem concentrations.

We report on a case of a 35-year-old man who died suddenly and unexpectedly due to a 4-fluoroisobutyrylfentanyl (4-FIBF) mono-intoxication. Pathological, toxicological and chemical investigations were conducted at the Netherlands Forensic Institute. A full three-cavity forensic pathological examination was performed according to international guidelines. Biological samples obtained during autopsy were comprehensively investigated for the presence of toxic substances using headspace gas chromatography (GC) with flame ionization detection, liquid chromatography-time-of-flight mass spectrometry (LC-TOF-MS), GC-MS, high-performance LC with diode array detection and LC-tandem MS (LC-MS-MS). The seized crystalline substance found next to the body was investigated using a presumptive color test, GC-MS, Fourier-transform infrared spectroscopy and nuclear magnetic resonance. Pathological investigation identified minor lymphocytic infiltrates in the heart, considered irrelevant for the cause of death. Toxicological analysis of the victims' blood indicated the presence of a fluorobutyrylfentanyl (FBF) isomer, with no other compounds detected. The FBF isomer was identified in the seized crystalline substance as 4-FIBF. 4-FIBF concentrations were quantified in femoral blood (0.030 mg/L), heart blood (0.12 mg/L), vitreous humor (0.067 mg/L), brain tissue (>0.081 mg/kg), liver tissue (0.44 mg/kg) and urine (approximately 0.01 mg/L). Based on the outcomes of the pathological, toxicological and chemical investigations, the cause of death of the deceased was attributed to a fatal 4-FIBF mono-intoxication. The presented case underlines the added value of a combined bioanalytical and chemical investigative approach to identify and subsequently quantify fentanyl isomers in postmortem cases. Furthermore, it demonstrates the importance of investigating the postmortem redistribution of novel fentanyl analogs to establish reference values and to subsequently allow for correct interpretation of cause of death analysis in future casework.

Tandem LC-MS Identification of Antitubercular Compounds in Zones of Growth Inhibition Produced by South African Filamentous Actinobacteria.

Novel antitubercular compounds are urgently needed to combat drug-resistant Mycobacterium tuberculosis (Mtb). Filamentous actinobacteria have historically been an excellent source of antitubercular drugs. Despite this, drug discovery from these microorganisms has fallen out of favour due to the continual rediscovery of known compounds. To increase the chance of discovering novel antibiotics, biodiverse and rare strains should be prioritised. Subsequently, active samples need to be dereplicated as early as possible to focus efforts on truly novel compounds. In this study, 42 South African filamentous actinobacteria were screened for antimycobacterial activity using the agar overlay method against the Mtb indicator Mycolicibacterium aurum under six different nutrient growth conditions. Known compounds were subsequently identified through extraction and high-resolution mass spectrometric analysis of the zones of growth inhibition produced by active strains. This allowed the dereplication of 15 hits from six strains that were found to be producing puromycin, actinomycin D and valinomycin. The remaining active strains were grown in liquid cultures, extracted and submitted for screening against Mtb in vitro. Actinomadura napierensis B60T was the most active sample and was selected for bioassay-guided purification. This resulted in the identification of tetromadurin, a known compound, but which we show for the first time to have potent antitubercular activity, with the MIC90s within the range of 73.7-151.6 nM against M. tuberculosis H37RvTin vitro under different test conditions. This shows that South African actinobacteria are a good source of novel antitubercular compounds and warrant further screening. It is also revealed that active hits can be dereplicated by HPLC-MS/MS analysis of the zones of growth inhibition produced by the agar overlay technique.

Overview of systematic toxicological analysis strategies and their coverage of substances in forensic toxicology.

Systematic toxicological analysis (STA) is the process of using an adequate analytical methodology to detect and identify as many potentially toxicologically relevant compounds as possible in biological samples. STA is an important part of everyday routine work within forensic toxicology, and several methods for STA have frequently been published and reviewed independently. However, the many drugs and other substances involved, as well as the constant emergence of new ones, may pose a major challenge in STA, which often demands a strategy involving multiple analytical methods in parallel. Such strategies have been published and evaluated less frequently despite their relevance in forensic toxicology. This mini-review briefly summarizes commonly applied methods for STA in forensic toxicology, including gas chromatography-mass spectrometry (GC-MS) and liquid chromatography-MS (LC-MS) methods, and highlights some of their potential pitfalls. Second, it provides an overview of previously reported strategies to conduct STA, including a presentation of the STA strategy applied in the authors' laboratory. This involves broad drug screening by LC-high-resolution MS, supported by targeted screening and quantification using LC-tandem MS, headspace (HS)-GC-MS, HS-GC-flame ionization detector and other complementary methods. The STA strategy aims to cover as many potentially relevant drugs as possible and seeks to reduce potential pitfalls arising in forensic casework. The review underlines that not every substance can be identified in all circumstances even with a comprehensive STA strategy.

Vitamin D status and prevalence of metabolic syndrome by race and Hispanic origin in US adults: findings from the 2007–2014 NHANES

Vitamin D status has been found to be inversely associated with metabolic syndrome (MetS) in some studies. Vitamin D status varies by race and ethnicity, and the association of MetS with vitamin D status in US adults and by race and Hispanic origin has not been evaluated extensively. We aimed to examine the associations between vitamin D status and MetS overall, and across race and Hispanic origin groups, in a nationally representative sample of US adults who participated in the NHANES from 2007 to 2014. The total sample included 8639 adults, ≥20 y of age. Serum vitamin D was measured using a standardized LC-tandem MS method and was categorized using data-driven tertiles. MetS was defined using measured waist circumference, triglycerides, HDL cholesterol, blood pressure, and fasting glucose. Multivariable logistic regression models were fitted [accounting for sociodemographic and lifestyle factors, dietary supplement use, and BMI (in kg/m2)] to examine the associations of serum vitamin D with MetS among adults overall, and by race and Hispanic origin. Serum vitamin D in the lowest tertile (≤56 nmol/L) was significantly associated with increased odds of MetS compared with the highest tertile (>77.9 nmol/L) (fully adjusted model OR: 1.85; 95% CI: 1.51, 2.27). Inverse associations were noted for all race-Hispanic origin groups: non-Hispanic white (NHW) (OR: 2.24; 95% CI: 1.67, 3.01), non-Hispanic black (OR: 1.56; 95% CI: 1.06, 2.29), and Hispanic (OR: 1.48; 95% CI: 1.03, 2.14) adults. Lower vitamin D status was significantly associated with MetS among US adults after adjusting for sociodemographic and lifestyle factors, dietary supplement use, and BMI. This finding was noted across all race and Hispanic origin groups, although the strength of the association varied, being strongest for NHW adults.

Individualized high dairy protein + walking program supports bone health in pregnancy: a randomized controlled trial

Pregnancy induces bone mineral mobilization, which may be further compromised if diet and physical activity are suboptimal. We aimed to determine the effects of a Nutrition+Exercise intervention during pregnancy on maternal calciotropic and bone biomarker profiles throughout pregnancy and the postpartum. In the Be Healthy in Pregnancy (BHIP) randomized controlled trial, 203 of 225 participants who consented to the bone health substudy were, randomly assigned at 12-17 weeks gestation to receive either usual care (control) or a structured and monitored Nutrition+Exercise plan (intervention) providing an individualized high dairy protein diet and a walking program throughout pregnancy. Maternal serum total procollagen type 1 N-terminal propeptide (P1NP; bone formation), C-terminal telopeptide of type I collagen (CTX; bone resorption), and insulin-like growth factor-1 (IGF-1) were measured by ELISA, and vitamin D metabolites by ultra-performance LC tandem MS at early and late pregnancy,6 mo postpartum, and in cord blood. In 187 participants completing all measures, significantly higher intakes were observed in the intervention than in the control group for total protein (P<0.0001), protein intake from dairy foods (P<0.0001), and calcium (P<0.0001), whereas vitamin D intake was similar between treatment groups in both the second and third trimesters. The intervention group had significantly lower serum CTX at end of pregnancy (mean±SD: 0.78±0.31 ng/mL; n=91 compared with 0.89±0.33 ng/mL; n=96, P=0.034) and in cord serum (0.58±0.13 ng/mL; n=31 compared with 0.69±0.18 ng/mL; n=22, P<0.025). Serum concentrations of P1NP rose significantly (P<0.02) from early pregnancy to 6 mo postpartum for the intervention group only. Serum 25-hydroxyvitamin D status was >50nmol/L for 97% of all participants. Higher maternal dietary protein and calcium intakes than usual care in concert with normal vitamin D status minimized bone resorption and maintained bone formation and may protect bone health during pregnancy.This trial was registered at clinicaltrials.gov as NCT01689961.

Emergency toxicology analysis – Method development for the straightforward determination of total 70 drugs and metabolites in human blood plasma by LC-MSn

Quantification of 70 emergency toxicology (ET) relevant analytes in human blood plasma using the Bruker Daltonik Toxtyper (TT) system. Rapid and comprehensive qualitative and quantitative methods are crucial in 24/7 ET to support the diagnosis and treatment of acute intoxications. This includes estimating their course and monitoring the effectiveness of possible detoxification. The aim of this study was the simultaneous qualitative and quantitative analysis of 64 drugs and six of their metabolites in human blood plasma by liquid chromatography-ion trap mass spectrometry (LC-MSn, Bruker Daltonik TT). Analytes included compound classes often requested in ET such as analgesics, anticonvulsants, antidepressants, antidiabetics, antiemetics, antihistamines, antihypertensives, benzodiazepines, or neuroleptics. A standard liquid-liquid extraction procedure was used for sample preparation followed by 1:5 dilution of the final extract (Caspar AC. Drug Test Anal 2019;11:102–111). Analyzes were performed using the TT system consisting of a Bruker amazon speed ion trap and a Thermo Fisher Dionex Ultimate 3000 LC system. Plasma concentrations were determined using an electronically stored five-point calibration. Validation was performed according to the European Medicines Agency (EMA) guideline on bioanalytical method validation and the recommendations of the Society of Toxicological and Forensic Chemistry (GTFCh) for ET quantification including selectivity, carry-over, accuracy, precision, and matrix effects. Finally, applicability was tested using 197 patient samples containing 37 different drugs and 5 of their metabolites. The method was selective for all analytes and no relevant carry-over and matrix effect could be observed. A linear and weighted calibration model could be used for all tested analytes. Accuracy and precision recommended for ET could be fulfilled for all tested analytes, except for aripiprazole, diphenhydramine, ketamine, melperone, norpethidine, nortilidine, olanzapine, metformin, pethidine, sertraline, and tilidine. The results after analysis of patient plasma were compared to those obtained by reference LC-tandem MS or gas chromatography-MS methods and data corresponded in case of 190 samples. The concentration levels in the remaining seven samples did not correspond to those determined by one of the reference methods. The present method provides a fast, robust, and reliable blood plasma screening for 70 analytes and an additional quantification of 59 analytes relevant in ET. Due to an electronically stored five-point calibration and a simple “push and print solution”, blood plasma levels of the analytes could be assessed straightforward. Furthermore, the applicability of the method by analyzing ET cases could be demonstrated.

Target Mechanism of Iturinic Lipopeptide on Differential Expression Patterns of Defense-Related Genes against Colletotrichum acutatum in Pepper.

Bacillus subtilis KB21 is an isolate with broad spectrum antifungal activity against plant pathogenic fungi. Our aim was to produce and purify antifungal lipopeptides via fermentation using B. subtilis KB21 and verify their antifungal mechanism against pepper anthracnose. When the KB21 strain was cultured in tryptic soy broth medium, the antifungal activity against pepper anthracnose correlated with biosurfactant production. However, there was no antifungal activity when cultured in Luria-Bertani medium. KB21 filtrates showed the highest degree of inhibition of mycelia (91.1%) and spore germination (98.9%) of Colletotrichum acutatum via increases in the biosurfactant levels. Using liquid chromatography-mass spectrometry (LC-MS) and LC-tandem MS (LC-MS/MS) analyses, the component with antifungal activity in the fermentation medium of the KB21 strain was determined to be the cyclic lipopeptide (CLP) antibiotic, iturin A. When the iturin fractions were applied to pepper fruits inoculated with conidia of C. acutatum, the lesion diameter and hyphal growth on the fruit were significantly suppressed. In addition, iturin CLP elevated the gene expression of PAL, LOX, and GLU in the treatments both with and without following fungal pathogens. Overall, the results of this study show that iturin CLPs from B. subtilis KB21 may be potential biological control agents for plant fungal diseases.

P06 Pharmacokinetics and renal accumulation of VRP-034

BackgroundNephrotoxicity is a dose-limiting factor for polymyxin B (PMB), a last-line therapy for MDR Gram-negative bacterial infections. The majority of the filtered PMB undergoes extensive tubular reabsorption leading to significant accumulation of the drug in tubular cells, causing renal tubular damage. We have previously reported that VRP-034, a novel PMB formulation, attenuates this damage (Roy et al.1). The objective of this study was to characterize the pharmacokinetics (PK) and renal accumulation of VRP-034 versus marketed PMB.Materials and methodsA total of 19 Sprague-Dawley rats were used in the study. For PK evaluation, 10 rats were administered a single subcutaneous dose (6 mg/kg) of either VRP-034 or marketed PMB. Serial plasma samples were collected up to 24 h and assayed for major PMB components (PMB B1, B1-I, B2) using a validated LC-tandem MS method. PK parameters were calculated using noncompartmental analysis. For evaluating renal deposition, nine rats (n = 3 each group) were administered VRP-034 or marketed PMB at a dose of 6 mg/kg/8 h for 48 h or normal saline (control). After 48 h, rats were euthanized and kidneys were excised by making a midline incision, washed with saline, homogenized in 1:9 w/v 0.1 M phosphate buffer (pH 7.4) and the homogenate was used for determining PMB concentration. All values are expressed as mean ± SEM.ResultsThe plasma pharmacokinetic parameters for both VRP-034 and marketed PMB were found similar [AUC0–24: 54.3 ± 4.7 μg/mL·h versus 51.5 ± 1.6 μg/mL·h; Cmax: 4.73 ± 0.6 μg/mL versus 5.02 ± 0.5 μg/mL; T½: 5.95 ± 0.5 h versus 5.6 ± 0.3 h]. In the renal deposition study, a 40% reduction (P<0.05) in renal deposition of PMB was found in VRP-034 treated rat kidneys compared with marketed PMB group after 48 h of treatment.ConclusionsThe results highlight that while the plasma PK parameters remained unchanged, a marked reduction in PMB renal deposition was observed in VRP-034 group and likely explains the previous reports of attenuated PMB-induced kidney injury with VRP-034.

Habitual intake of dietary advanced glycation end products is not associated with generalized microvascular function—the Maastricht Study

ABSTRACTBackgroundEndogenously formed advanced glycation end products (AGEs) may be important drivers of microvascular dysfunction and the microvascular complications of diabetes. AGEs are also formed in food products, especially during preparation methods involving dry heat.ObjectivesWe aimed to assess cross-sectional associations between dietary AGE intake and generalized microvascular function in a population-based cohort.MethodsIn 3144 participants of the Maastricht Study (mean ± SD age: 60 ± 8 y, 51% men) the dietary AGEs Nε-(carboxymethyl)lysine (CML), Nε-(1-carboxyethyl)lysine (CEL), and Nδ-(5-hydro-5-methyl-4-imidazolon-2-yl)-ornithine (MG-H1) were estimated using the combination of our ultra-performance LC–tandem MS dietary AGE database and an FFQ. Microvascular function was determined in the retina as flicker light–induced arteriolar and venular dilation and as central retinal arteriolar and venular equivalents, in plasma as a z score of endothelial dysfunction biomarkers (soluble vascular adhesion molecule 1 and soluble intracellular adhesion molecule 1, soluble E-selectin, and von Willebrand factor), in skin as the heat-induced skin hyperemic response, and in urine as 24-h albuminuria. Associations were evaluated using multiple linear regression adjusting for demographic, cardiovascular, lifestyle, and dietary factors.ResultsOverall, intakes of CML, CEL, and MG-H1 were not associated with the microvascular outcomes. Although higher intake of CEL was associated with higher flicker light–induced venular dilation (β percentage change over baseline: 0.14; 95% CI: 0.02, 0.26) and lower plasma biomarker z score (β: −0.04 SD; 95% CI: −0.08, −0.00 SD), the effect sizes were small and their biological relevance can be questioned.ConclusionsWe did not show any strong association between habitual intake of dietary AGEs and generalized microvascular function. The contribution of dietary AGEs to generalized microvascular function should be further assessed in randomized controlled trials using specifically designed dietary interventions.

Determination of cannabinoids in hemp oil based cosmetic products by LC-tandem MS.

Cosmetic products containing hemp seed oil as permitted raw materials required the specific compound delta-9-tetrahydrocannabinol (THC) below 10 μg/g. THC was the main psychoactive constituent of cannabis. Since hemp seed oil became an increasingly popular ingredient in cosmetics over the last few years, an efficient and reliable analytical method for THC and other cannabinoids in cannabis-infused cosmetic products was in need. A liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for the determination of delta-9-tetrahydrocannabinol (THC), cannabidiol (CBD), and cannabinol (CBN) in hemp seed oil based cosmetic products was developed. Method validation was performed by fertilizing blank samples with analytes and internal standards (THC-d3, CBD-d3, and CBN-d3). Chromatographic method utilized a Xbridge BEH Shield RP18 column with gradient elution containing 10 mM ammonium formate in water and methanol provided successful separation of THC, CBD, and CBN in cosmetic matrix. The combination of MS detection in positive electrospray ionization (ESI) and multiple reaction monitoring (MRM) mode offered rapid run time 13 minutes with limit of quantification (LOQ) of 0.05 μg/g. The intra- and inter-day recoveries were 79.23–114.04% and 83.55–111.61% with spiking levels ranged between 0.05 μg/g and 0.5 μg/g, respectively. Surveillance results of 90 cosmetic products showed 22, 34, and 5 products containing THC (0.06–1777 μg/g), CBD (0.47–37217 μg/g), and CBN (2.2–25.2 μg/g), respectively. This validated method offered accurate, reliable, and fast way for the determination of drug contaminations including THC, CBD, and CBN in cosmetics. The surveillance results for commercial cosmetic products purchased in Taiwan between 2018–2020 provided valuable background references for THC, CBD, and CBN in hemp seed oil based cosmetic products, and could be used for administration purpose.

Pharmacokinetic study of Ninjin'yoeito: Absorption and brain distribution of Ninjin'yoeito ingredients in mice

Ethnopharmacological relevanceNinjin'yoeito (NYT), a Japanese traditional Kampo medicine, has been reported to exert various clinical benefits such as relief from fatigue, malaise, anorexia, frailty, sarcopenia, and cognitive dysfunction. Recently, some review articles described the pharmacological effects of NYT and additionally indicated the possibility that multiple ingredients in NYT contribute to these effects. However, pharmacokinetic data on the ingredients are essential in addition to data on their pharmacological activities to accurately determine the active ingredients in NYT. Aim of the studyThis study assessed the in vivo pharmacokinetics of NYT using mice. Materials and methodsTarget liquid chromatography–mass spectrometry (LC–MS) and wide target LC–MS or LC–tandem MS of NYT ingredients in plasma and the brain after oral administration of NYT were performed. Imaging MS was performed to investigate the detailed brain distributions of NYT ingredients. ResultsThe concentrations of 13 ingredients in plasma and schizandrin in the brain were quantified via target LC–MS, and the wide target analysis illustrated that several ingredients are absorbed into blood and transported into the brain. Imaging MS revealed that schizandrin was homogenously dispersed in the NYT-treated mouse brain. ConclusionThese results should be useful for clarifying the active ingredients of NYT and their mechanisms of actions.

Transcriptional, hormonal, and metabolic changes in susceptible grape berries under powdery mildew infection.

Grapevine (Vitis vinifera) berries are extremely sensitive to infection by the biotrophic pathogen Erysiphe necator, causing powdery mildew disease with deleterious effects on grape and wine quality. The combined analysis of the transcriptome and metabolome associated with this common fungal infection has not been previously carried out in any fruit. In order to identify the molecular, hormonal, and metabolic mechanisms associated with infection, healthy and naturally infected V. vinifera cv. Carignan berries were collected at two developmental stages: late green (EL33) and early véraison (EL35). RNA sequencing combined with GC-electron impact ionization time-of-flight MS, GC-electron impact ionization/quadrupole MS, and LC-tandem MS analyses revealed that powdery mildew-susceptible grape berries were able to activate defensive mechanisms with the involvement of salicylic acid and jasmonates and to accumulate defense-associated metabolites (e.g. phenylpropanoids, fatty acids). The defensive strategies also indicated organ-specific responses, namely the activation of fatty acid biosynthesis. However, defense responses were not enough to restrict fungal growth. The fungal metabolic program during infection involves secretion of effectors related to effector-triggered susceptibility, carbohydrate-active enzymes and activation of sugar, fatty acid, and nitrogen uptake, and could be under epigenetic regulation. This study also identified potential metabolic biomarkers such as gallic, eicosanoic, and docosanoic acids and resveratrol, which can be used to monitor early stages of infection.

AlphaTims: Indexing Trapped Ion Mobility Spectrometry–TOF Data for Fast and Easy Accession and Visualization

High-resolution MS-based proteomics generates large amounts of data, even in the standard LC–tandem MS configuration. Adding an ion mobility dimension vastly increases the acquired data volume, challenging both analytical processing pipelines and especially data exploration by scientists. This has necessitated data aggregation, effectively discarding much of the information present in these rich datasets. Taking trapped ion mobility spectrometry (TIMS) on a quadrupole TOF (Q-TOF) platform as an example, we developed an efficient indexing scheme that represents all data points as detector arrival times on scales of minutes (LC), milliseconds (TIMS), and microseconds (TOF). In our open-source AlphaTims package, data are indexed, accessed, and visualized by a combination of tools of the scientific Python ecosystem. We interpret unprocessed data as a sparse four-dimensional matrix and use just-in-time compilation to machine code with Numba, accelerating our computational procedures by several orders of magnitude while keeping to familiar indexing and slicing notations. For samples with more than six billion detector events, a modern laptop can load and index raw data in about a minute. Loading is even faster when AlphaTims has already saved indexed data in an HDF5 file, a portable scientific standard used in extremely large-scale data acquisition. Subsequently, data accession along any dimension and interactive visualization happens in milliseconds. We have found AlphaTims to be a key enabling tool to explore high-dimensional LC-TIMS-Q-TOF data and have made it freely available as an open-source Python package with a stand-alone graphical user interface at https://github.com/MannLabs/alphatims or as part of the AlphaPept ‘ecosystem’.