Liquid Chromatography-mass Spectrometry Assay Research Articles

Therapeutic drug monitoring of osimertinib in EGFR mutant non-small cell lung cancer by dried blood spot and plasma collection: A pilot study.

Therapeutic drug monitoring (TDM) has led to significant improvements in individualized medical care, although its implementation in oncology has been limited to date. Tyrosine kinase inhibitors (TKIs) are a group of therapies for which TDM has been suggested. Osimertinib is one such therapy used in the treatment of epidermal growth factor receptor (EGFR) mutation-driven lung cancer. Herein, we describe a prospective pilot study involving 21 patients on osimertinib primarily as a preliminary evaluation of drug levels in a real-world setting. Concentrations of the drug and its primary metabolites were measured with a validated liquid chromatography-mass spectrometry (LC-MS) assay across serial timepoints. As part of this study, inter-individual variability by dose and ethnicity as well as intra-individual variability across timepoints are explored. Furthermore, we attempted to validate dried blood spot (DBS)-based quantitation as an accurate alternative to plasma quantitation. Successful quantitation of osimertinib and primary metabolites was achieved for our subjects. Compound plasma levels were highly correlated to DBS levels. There was no significant difference in concentrations with ethnicity or dosing or intra-individual variability across timepoints. As such, we demonstrate that TDM for osimertinib is practical for future trials. We also validated the use of DBS as an alternative to conventional quantitation for exploration of TDM for osimertinib in larger trials and for other targeted therapies.

Methods for research on structures and functions of traditional Chinese medicine proteins

Traditional Chinese medicine proteins(TCMPs) not only have nutritional values and biological activities but also serve as key enzymes in the synthesis of pharmacodynamic components in traditional Chinese medicines. They play a role in the synthesis of pharmacodynamic components by regulating biosynthesis and selective synthesis pathways and controlling drug quality and stability. The recent years have witnessed great progress in the research on the structures and functions of proteins using various methods and technologies. However, the research on the structures and functions of TCMPs lags behind. Therefore, it is urgent to study the structures and functions of TCMPs using modern means to promote the discovery of innovative drugs based on TCMPs and clarify the synthesis pathways of pharmacodynamic components. This study introduces the latest techniques for studying protein structures and functions, including spectroscopy, mass spectrometry, nuclear magnetic resonance, X-ray crystal diffraction, microscopy, and structure prediction. Furthermore, this paper introduces the methods for protein functional studies, including liquid chromatography-mass spectrometry, co-immunoprecipitation, yeast two-hybrid, and pull-down assay. By systematically reviewing these techniques and methods, this paper provides technical references for the structural identification and functional studies of TCMPs, with the aim of promoting the in-depth exploration of the structures and functions of TCMPs.

Correlation between serum tryptophan metabolism and treatment efficacy of dapoxetine in patients with premature ejaculation: A pilot study.

Primary premature ejaculation (PPE) is a common male neurobiological disorder. Currently, there is consensus that the impairment in central serotonin (5-HT) neurotransmission constitutes a key pathogenic factor in PPE. Selective serotonin reuptake inhibitors (SSRIs) serve as the primary pharmacological intervention; however, a comprehensive elucidation of their mechanism of action remains incomplete. Owing to significant individual variability in efficacy, SSRIs exhibit a high discontinuation rate. Hence, there is an urgent need to address the selection of SSRIs for PPE treatment. This study aims to investigate the characteristics of tryptophan (TRP) metabolism in patients with PPE and to assess its influence on the efficacy of SSRIs. The exploratory study included a total of 16 patients with PPE and 16 control subjects who were healthy men without any sexual dysfunction. Upon enrollment in the study, all participants underwent a thorough medical history review and physical examination. Subsequently, their serum levels of TRP, its metabolites, large neutral amino acids (LNAAs), and metabolite ratios were assessed using a liquid chromatography-mass spectrometry (LC-MS) assay. After a period of 4weeks of dapoxetine treatment, all patients with PPE underwent reassessment using the Premature Ejaculation Diagnostic Tool (PEDT) score and intravaginal ejaculatory latency time (IELT) test. The ratio of serum TRP to other LNAAs (TRP/LNAAs) in patients with PPE was found to be significantly lower compared to the control group (P<0.05). Conversely, the ratio of kynurenine to TRP (KYN/TRP) was observed to be significantly higher in the PPE patients compared to the control group (P<0.05). Including the serum TRP/LNAAs ratio and KYN/TRP ratio in the prediction model yielded the highest prediction efficiency for PPE. There was a significant negative correlation between the ratio of TRP/LNAAs before the treatment and the IELT after 4weeks of the treatment. Additionally, there was a significant positive correlation observed between the ratio of TRP/LNAAs before the treatment and the PEDT score after 4weeks of the treatment. This study demonstrates that the reduction in the TRP/LNAAs ratio and the elevation of the KYN/TRP ratio are significant characteristics associated with PPE. These findings suggest that diminished tryptophan availability in the brain and the activation of the kynurenine (KYN) pathway may play a role in the pathogenesis of PPE. The TRP/LNAAs ratio has potential as a reliable indicator of central serotonin (5-HT) levels. Considering the TRP/LNAAs ratio when selecting SSRIs for the treatment of PPE may enhance the response rate of this medication.

Cross-Validation of Metabolic Phenotypes in SARS-CoV-2 Infected Subpopulations Using Targeted Liquid Chromatography-Mass Spectrometry (LC-MS).

To ensure biological validity in metabolic phenotyping, findings must be replicated in independent sample sets. Targeted workflows have long been heralded as ideal platforms for such validation due to their robust quantitative capability. We evaluated the capability of liquid chromatography-mass spectrometry (LC-MS) assays targeting organic acids and bile acids to validate metabolic phenotypes of SARS-CoV-2 infection. Two independent sample sets were collected: (1) Australia: plasma, SARS-CoV-2 positive (n = 20), noninfected healthy controls (n = 22) and COVID-19 disease-like symptoms but negative for SARS-CoV-2 infection (n = 22). (2) Spain: serum, SARS-CoV-2 positive (n = 33) and noninfected healthy controls (n = 39). Multivariate modeling using orthogonal projections to latent structures discriminant analyses (OPLS-DA) classified healthy controls from SARS-CoV-2 positive (Australia; R2 = 0.17, ROC-AUC = 1; Spain R2 = 0.20, ROC-AUC = 1). Univariate analyses revealed 23 significantly different (p < 0.05) metabolites between healthy controls and SARS-CoV-2 positive individuals across both cohorts. Significant metabolites revealed consistent perturbations in cellular energy metabolism (pyruvic acid, and 2-oxoglutaric acid), oxidative stress (lactic acid, 2-hydroxybutyric acid), hypoxia (2-hydroxyglutaric acid, 5-aminolevulinic acid), liver activity (primary bile acids), and host-gut microbial cometabolism (hippuric acid, phenylpropionic acid, indole-3-propionic acid). These data support targeted LC-MS metabolic phenotyping workflows for biological validation in independent sample sets.

Elevated KDM4D Expression in Pterygium: Impact and Potential Inhibition by Lycium Barbarum Polysaccharide.

Purpose: This study aimed to explore the effects of elevated KDM4D expression and potential therapeutic effects of Lycium barbarum polysaccharide (LBP) on pterygium. Methods: The expression levels of KDM4D in the primary pterygium (n = 29) and normal conjunctiva (n = 14) were detected by immunohistochemistry. The effects of KDM4D on pterygium fibroblasts were detected by the CCK-8 assay, liquid chromatography-mass spectrometry assay, flow cytometry, and scratch wound healing assay. The relative expression of KDM4D in pterygium fibroblasts stimulated by interleukin (IL)-1β, IL-6, IL-8, and LBP was detected by quantitative real-time PCR and Western blot. The effects of LBP on pterygium fibroblasts were detected using flow cytometry and scratch wound healing assays. Results: The expression level of KDM4D in pterygium was higher than that in normal conjunctiva. KDM4D increased the cell viability of pterygium fibroblasts. The differentially expressed genes identified in the LM-MS assay enriched in "actin filament organization" and "apoptosis." KDM4D promoted migration and inhibited apoptosis of pterygium fibroblasts in vitro. Inflammatory cytokines, including IL-1β, IL-6, and IL-8, enhanced the expression of KDM4D in pterygium fibroblasts. LBP inhibited the expression of KDM4D in pterygium fibroblasts and decreased their cell viability. Moreover, LBP attenuated the KDM4D effects on migration and apoptosis of pterygium fibroblasts. Conclusions: Elevated KDM4D expression is a risk factor for pterygium formation. LBP inhibits the expression of KDM4D in pterygium fibroblasts and may be a potential drug for delaying pterygium development.

Therapeutic drug monitoring of clozapine in human serum by high-throughput paper spray mass spectrometry

IntroductionMonitoring the atypical antipsychotic drug clozapine is crucial to ensure patient safety. This article showcases a high-throughput analytical method for measuring clozapine and its primary metabolite norclozapine (N-desmethylclozapine) in serum using paper spray mass spectrometry (PS-MS). ObjectivesThis study aimed to assess the viability of a PS-MS method for the rapid measurement of clozapine and norclozapine in human serum samples as an alternative to liquid chromatography mass spectrometry (LC-MS). MethodsSerum samples were processed by protein precipitation followed by deposition of the supernatant containing labelled internal standards onto paper spray substrates mounted in cartridges. Analytes were then analyzed using a triple quadrupole mass spectrometer equipped with a commercial paper spray ionization source. The results obtained from the patient samples were compared to those from a validated LC-MS assay. ResultsPS-MS calibrations for clozapine and norclozapine were linear (R2 > 0.99) over five days. Between-run precision was below 8 %, and within-run precision did not exceed 10 %. When compared to a validated LC-MS method, the mean bias for 39 patient samples was −9% for clozapine and −1% for norclozapine, with no outliers. Mass spectrometry ion ratio comparisons indicated no interference for patient samples above the lower limit of quantification. There was less than 7 % change in the measured concentrations of both analytes over five days for samples dried on paper substrates. Notably, virtually no maintenance of the MS source was required during this study. ConclusionThis study illustrates the potential of PS-MS for serum drug monitoring in the clinical laboratory.

An improved workflow for the development of MS-compatible liquid chromatography assay purity and purification methods by using automated LC Screening instrumentation and in silico modeling.

The development of liquid chromatography UV and mass spectrometry (LC-UV-MS) assays in pharmaceutical analysis is pivotal to improve quality control by providing critical information about drug purity, stability, and presence and identity of byproducts and impurities. Analytical method development of these assays is time-consuming, which often causes it to become a bottle neck in drug development and poses a challenge for process chemists to quickly improve the chemistry. In this study, a systematic and efficient workflow was designed to develop purity assay and purification methods for a wide range of compounds including peptides, proteins, and small molecules with MS-compatible mobile phases (MP) by using automated LC screening instrumentation and in silico modeling tools. Initial LC MPs and chromatography column screening experiments enabled quick identification of conditions which provided the best resolution in the vicinity of the target compounds, which is further optimized using computer-assisted modeling (LC Simulator from ACD/Labs). The experimental retention times were in good agreement with the predicted retention times from LC Simulator (ΔtR < 7%). This workflow presents a practical workflow to significantly expedite the time needed to develop optimized LC-UV-MS methods, allowing for a facile, automatic method optimization and reducing the amount of manual work involved in developing new methods during drug development.

Comparison of colorimetric, fluorometric, and liquid chromatography-mass spectrometry assays for acetyl-coenzyme A

Acetyl-Coenzyme A is a central metabolite in catabolic and anabolic pathways as well as the acyl donor for acetylation reactions. Multiple quantitative measurement techniques for acetyl-CoA have been reported, including commercially available kits. Comparisons between techniques for acetyl-CoA measurement have not been reported. This lack of comparability between assays makes context-specific assay selection and interpretation of results reporting changes in acetyl-CoA metabolism difficult. We compared commercially available colorimetric ELISA and fluorometric enzymatic-based kits to liquid chromatography-mass spectrometry-based assays using tandem mass spectrometry (LC-MS/MS) and high-resolution mass spectrometry (LC-HRMS). The colorimetric ELISA kit did not produce interpretable results even with commercially available pure standards. The fluorometric enzymatic kit produced comparable results to the LC-MS-based assays depending on matrix and extraction. LC-MS/MS and LC-HRMS assays produced well-aligned results, especially when incorporating stable isotope-labeled internal standards. In addition, we demonstrated the multiplexing capability of the LC-HRMS assay by measuring a suite of short-chain acyl-CoAs in a variety of acute myeloid leukemia cell lines and patient cells.

Screening the NCI diversity set V for anti-MRSA activity: cefoxitin synergy and LC-MS/MS confirmation of folate/thymidine biosynthesis inhibition.

New antibacterial agents and agent combinations are urgently needed to combat antimicrobial resistance. A multidimensional chemical library screening strategy was used to identify compounds in the National Cancer Institute (NCI) diversity set V library (1,593 compounds) with anti-methicillin-resistant Staphylococcus aureus (MRSA) activity. In this effort, library compounds were screened for anti-MRSA activity in both their original [un-metabolized (UM)] and human liver microsome-metabolized [post-metabolized (PM)] forms and in the absence and presence of sub-minimum inhibitory concentration (MIC) levels of cefoxitin. This strategy allows for the identification of intrinsically active agents, agents with active metabolites, and agents that can act synergistically with cefoxitin. Sixteen UM compounds with MICs ≤ 12.5 µM were identified. No agents with substantially enhanced activity after microsomal metabolism were found. Several agents showed significant apparent synergy with cefoxitin, and checkerboard assays were used to confirm synergy for four of these (celastrol, porfiromycin, 4-quinazolinediamine, and teniposide). A follow-up comparative screen in the absence and presence of 4-µM thymidine was used to identify three agents as likely folate/thymidine biosynthesis inhibitors. A liquid chromatography-mass spectrometry (LC-MS/MS) assay for deoxythymidine triphosphate (dTTP) was used to confirm these three as suppressing dTTP biosynthesis in MRSA. Bactericidal vs bacteriostatic activity was also evaluated. This study further demonstrates the utility of comparative library screening to identify novel bioactive agents with interesting synergies and biological activities. The identification of several folate/thymidine biosynthesis inhibitors from this small screen indicates that this pathway is a viable target for new drug discovery efforts. IMPORTANCE New antibacterial agents are urgently needed to counter increasingly resistant bacteria. One approach to this problem is library screening for new antibacterial agents. Library screening efforts can be improved by increasing the information content of the screening effort. In this study, we screened the National Cancer Institute diversity set V against methicillin-resistant Staphylococcus aureus (MRSA) with several enhancements. One of these is to screen the library before and after microsomal metabolism as means to identify potential active metabolites. A second enhancement is to screen the library in the absence and presence of sub-minimum inhibitory concentration levels of another antibiotic, such as cefoxitin in this study. This identified four agents with synergistic activity with cefoxitin out of 16 agents with good MRSA activity alone. Finally, active agents from this effort were counter-screened in the presence of thymidine, which quickly identified three folate/thymidine biosynthesis inhibitors, and also screened for bactericidal vs bacteriostatic activity.

The HNF4A-CHPF pathway promotes proliferation and invasion through interactions with MAD1L1 in glioma.

Chondroitin polymerizing factor (CHPF) is an important glycosyltransferases that participates in the biosynthesis of chondroitin sulfate (CS). Our previous study showed that silencing CHPF expression inhibited glioma cell proliferation in vitro, but the molecular mechanisms by which CHPF contributes to development of glioma have not been characterized. In this study, we found that CHPF was up-regulated in glioma tissues and was positively correlated with malignant clinical pathological characteristics of patients with glioma. Silencing CHPF expression inhibited proliferation, colony formation, migration, and cell cycle of glioma cells. Moreover, silencing CHPF suppressed glioma malignance in vivo. Immunoprecipitation, co-immunoprecipitation, GST pulldown, and liquid chromatography-mass spectrometry (LC-MS/MS) assays were used to verify the interaction between CHPF and Mitotic arrest deficient 1-like 1 (MAD1L1). In addition, Chromatin Immunoprecipitation (ChIP)-PCR analysis showed that HNF4A bound to the CHPF promoter region, which indicated that the transcription factor hepatocyte nuclear factor 4A (HNF4A) could regulate the expression of CHPF in glioma cells.

FRI176 Integrated Profiling Analysis Of Serum Steroids And Catecholamines In A Single Run With LC-MS

Abstract Disclosure: J. Noh: None. C. Lee: None. S. Park: None. C. Ahn: None. S. Myung: None. J. Kim: None. M. Choi: None. Adrenal steroids and catecholamines are associated with the response against biochemical stress and adrenal disorders. Metabolic alteration of these hormones could be quantitatively evaluated in diagnostic process, but both adrenal cortex and medullary hormones have been measured in different analytical platforms. To increase the bio-analytical validity, liquid chromatography-mass spectrometry (LC-MS) based simultaneous profiling of 29 steroids and 6 catecholamines was developed. After alkyloxycarbonylation with isobutyl chloroformate, catecholamines and steroids were extracted simultaneously in liquid-liquid extraction. The limit of quantification ranged from 0.1 to 25 ng/mL with extraction recoveries of 78.5-97.0%, while the precision and accuracy were 4.8-7.9% and 98.0-110.9%, respectively. The validated LC-MS assay was then applied to serum samples obtained from 20 individual patients with primary aldosteronism (PA), Cushing’s syndrome (CS) and pheochromocytoma and paraganglioma (PPGL). The significantly increased serum levels of 18-hydroxycorticosterone (p &amp;lt; 0.005), corticosterone (p &amp;lt; 0.05), and 18-hydroxycortisol (p &amp;lt; 0.002) were observed in PA group, while PPGL group showed decreased level of 11-deoxycortisol (p &amp;lt; 0.007). Higher levels of glucocorticoids including 21-deoxycortisol (p &amp;lt; 0.002), 6β-hydroxycortisol (p &amp;lt; 0.001), and tetrahydrocortisone (p &amp;lt; 0.001) and lower levels of androgens including DHEA (p &amp;lt; 0.00001) and DHEA sulfate (p &amp;lt; 0.03) were detected in CS group. Catecholamines, such as norepinephrine (p &amp;lt; 0.0001) and normetanephrine (p &amp;lt; 0.0001), were significantly increased in PPGL patients. The present novel assay was successfully applied to discriminate three different adrenal diseases in a single analytical run. Presentation: Friday, June 16, 2023

An exploratory study on the possible association of serum etonogestrel concentrations with mood concerns and symptoms among contraceptive implant users

ObjectiveTo evaluate if inter-individual variability in serum etonogestrel (ENG) concentrations accounts for variability in mood-related side effects among ENG implant users. Study DesignParticipants underwent a single-time blood draw for measurement of serum ENG concentrations using a liquid-chromatography mass-spectrometry assay, and completed a questionnaire at enrollment that retrospectively assessed mood-related side effects during the period of implant use. For a subset of participants, Patient Health Questionnaire-9 (PHQ-9) scores, obtained for other clinical purposes, were also compared. We used independent medians tests and linear regression to evaluate associations between mood symptoms and serum ENG concentrations as our primary outcome. ResultsAmong 900 enrolled participants, 34% (306/900) reported mood changes on the baseline questionnaire. Of these, 31 (3.4%) participants also had documented PHQ-9 scores. Serum ENG concentrations (median 126.9 pg/mL [range 39.4–695.1]) were not associated with reported mood changes on the questionnaire (p = 0.19) or on the PHQ-9 (β = 0.00, 95% CI −0.03, 0.03). ConclusionPharmacokinetic variability does not explain the inter-individual variability in mood-related side effects among ENG implant users. ImplicationsMood-related side effects and altered mental health metrics are commonly reported by etonogestrel contraceptive implant users but demonstrate wide inter-individual variability. Individual differences in serum drug levels do not appear to account for this variability in mood-related side effects, and so future research should focus on novel personal factors.

A-049 Inter-assay Variation of Insulin-like Growth Factor-1 Remains an Issue

Abstract Background Human insulin-like growth factor-1 (IGF-1) is the main mediator of the somatotropic effects of growth hormone (GH). Accurate measurement of IGF-1 is required for the diagnosis and management of GH secretion disorders. Several commercial vendors and laboratories have standardized their IGF-1 assays to the WHO International Standard 02/254. This standardization does not prohibit the existence of significant inter-assay variability in IGF-1 measurements. Such concentration variance can lead to inaccurate patient case decision making. The IGF-1 testing performed in our laboratory was recently migrated from an Immulite 2000 platform to a CobasPro system. This shift in vendor necessitated an investigation of the inter-assay variability between these standardized tests. This relative variance was established through a validation of the analytical performance of the CobasPro IGF-1 assay. Methods Reportable range, intra-day precision and accuracy of IGF-1 measurements using the CobasPro (Roche Diagnostics) system were determined. Patient correlation studies between the CobasPro and an Immulite 2000 (Siemens Healthcare Diagnostics) instrument was also performed. The concentration of IGF-1 in a subset of these correlation study specimens was also quantified by liquid chromatography mass spectrometry (LC-MS) assays from two different reference laboratories. Results The CobasPro assay had a verified linear range of 7–1443 ng/mL (R2 = 0.9998, slope = 0.9915). At IGF-1 concentrations of 54 and 345 ng/mL the intra-day precision (%CV, N = 10) of the CobasPro assay did not exceed 0.9%. Patient specimen correlations between the CobasPro and Immulite 2000 assays showed significant bias (Deming regression: y = 1.543x − 48.61, N = 128, R = 0.9641). This method bias was exacerbated at Immulite 2000 derived IGF-1 concentrations &amp;gt;100 ng/mL (Deming regression: y = 1.662x − 84.29, N = 93, R = 0.9492, CobasPro range = 77–910 ng/mL, Immulite 2000 range = 103–539 ng/mL). Patient specimen correlations between the CobasPro and LC-MS assay from reference laboratory A showed a positive bias (Deming regression: y = 1.375x − 64.5, N = 19, R = 0.9210). An analogous correlation with reference laboratory B demonstrated a higher level of concordance (Deming regression: y = 1.005x + 10.2, N = 16, R = 0.9499). The CobasPro and Immulite 2000 assays both produced acceptable result when challenged by multiple external quality assurance (EQA) specimens provided by the College of American Pathologists (CAP). However, the observed relative trends in IGF-1 concentrations from these challenges were opposite to our observations with authentic patient specimens. The CobasPro assay consistently produced lower IGF-1 concentrations relative to the Immulite 2000 assay for these EQA specimens. Conclusion The CobasPro IGF-1 assay offered acceptable analytical performance. Although both assays were traceable to a reference standard, a significant positive concentration bias was observed between the CobasPro and Immulite 2000 assays. Standardization efforts have not eliminated inter-assay IGF-1 variability. A careful evaluation of the impact of this variance on the diagnosis and management of GH deficiency and acromegaly must be considered when IGF-1 testing platforms are changed.

ACVRL1 drives resistance to multitarget tyrosine kinase inhibitors in colorectal cancer by promoting USP15-mediated GPX2 stabilization

BackgroundMultitarget tyrosine kinase inhibitors (mTKIs) such as Regorafenib and Sorafenib have already been approved for the treatment of many solid tumours. However, the efficacy of mTKIs in colorectal cancer (CRC) is limited; the underlined mechanism remains largely elusive. Our study was aimed to find out the resistance mechanism of mTKIs in CRC.MethodsRNA sequencing was used to identify the expression of Activin A receptor-like type 1 (ACVRL1) under the treatment of mTKIs. Gain/loss-of-function experiments were performed to assess the biological function of ACVRL1 in resistance to mTKIs. The underlying mechanisms of ACVRL1-mediated mTKI resistance were investigated by using liquid chromatography-mass spectrometry assays (LC-MS), co-immunoprecipitation assays (Co-IP), chromatin immunoprecipitation assays, ubiquitination assays, dual luciferase reporter assays, etc.ResultsRNA sequencing identified the activation of ACVRL1 under the treatment of mTKIs in CRC cells. ACVRL1 knockdown and overexpression significantly affects the sensitivity of CRC cells to mTKIs both in vitro and vivo. Mechanistically, we found the β-catenin/TCF-1-KCNQ1OT1/miR-7-5p axis mediated the activation of ACVRL1.Furthermore, LC-MS assays indicated the interaction between ACVRL1 and glutathione peroxidase 2(GPX2) protein. IP assay defined ACVRL1 truncation (282–503aa) could be responsible for interacting with GPX2, and rescue experiments with ACVRL1 truncations confirmed the importance of this interaction in driving mTKI resistance. Co-IP assays confirmed that ACVRL1 associates with ubiquitin-specific peptidase 15(USP15) which directly deubiquinates GPX2 at the K187(K, lysine) site, leading to the accumulation of GPX2 protein. Rescue experiments performed with the lysine mutants in GPX2 CRISPR knockout cell model confirmed the importance of GPX2 K187 mutant. As a result, the increased ROS clearance and decreased cell apoptosis eventually lead to mTKI resistance in CRC.ConclusionsOur results demonstrate that the Wnt/β-catenin/KCNQ1OT1/miR-7-5p/ACVRL1/GPX2 biological axis plays a vital role in CRC, targeting which may be an effective approach for overcoming mTKI resistance.

A supervised machine-learning approach for the efficient development of a multi method (LC-MS) for a large number of drugs and subsets thereof: focus on oral antitumor agents.

Accumulating evidence argues for a more widespread use of therapeutic drug monitoring (TDM) to support individualized medicine, especially for therapies where toxicity and efficacy are critical issues, such as in oncology. However, development of TDM assays struggles to keep pace with the rapid introduction of new drugs. Therefore, novel approaches for faster assay development are needed that also allow effortless inclusion of newly approved drugs as well as customization to smaller subsets if scientific or clinical situations require. We applied and evaluated two machine-learning approaches i.e.,a regression-based approach and an artificial neural network (ANN) to retention time (RT) prediction for efficient development of a liquid chromatography mass spectrometry (LC-MS) method quantifying 73 oral antitumor drugs (OADs) and five active metabolites. Individual steps included training, evaluation, comparison, and application of the superior approach to RT prediction, followed by stipulation of the optimal gradient. Both approaches showed excellent results for RT prediction (mean difference ± standard deviation: 2.08 % ± 9.44 % ANN; 1.78 % ± 1.93 % regression-based approach). Using the regression-based approach, the optimum gradient (4.91 % MeOH/min) was predicted with a total run time of 17.92 min. The associated method was fully validated following FDA and EMA guidelines. Exemplary modification and application of the regression-based approach to a subset of 14 uro-oncological agents resulted in a considerably shortened run time of 9.29 min. Using a regression-based approach, a multi drug LC-MS assay for RT prediction was efficiently developed, which can be easily expanded to newly approved OADs and customized to smaller subsets if required.

A validated method for detection of cannflavins in hemp extracts

A selective and sensitive liquid chromatography mass spectrometry assay was developed for the detection of cannflavin A, B, and C in hemp extract specimens. A deuterated analog cannabidiol-D3 was used as the internal standard and the isocratic method used a mobile phase consisting of acetonitrile and water with 0.1 % formic acid [83:17]. Detection was carried out by electrospray positive ionization in single-ion monitoring mode through a C-18 analytical column. The assay (total run time <20 min) had excellent linearity and a lower limit of quantification of 0.5 μg/mL and a limit of detection of 0.25 μg/mL with a 10 μL injection. The method possessed suitable measures of stability, sensitivity, and selectivity for detecting cannflavins in several specimen types. The method was successfully applied to the analysis of samples of cannflavin release from prototype topical formulations.

Simultaneous quantitation of ketamine, norketamine and dehydronorketamine in human milk using a novel ultra high-performance liquid chromatography-mass spectrometry (UPLC-MS/MS) assay

There is a paucity of data on the transfer of ketamine from maternal blood into human milk. Quantification of ketamine in human milk provides information about the potential exposure of the infant to ketamine and its metabolites from the mother during lactation. A highly specific, reproducible, and sensitive UPLC-MS/MS based analytical method was developed and validated for the quantitation of ketamine and its metabolites (norketamine and dehydronorketamine) in human milk. Samples were subjected to a simple protein precipitation and ketamine-d4 and norketamine-d4 were used as internal standards. Separation of the analytes was achieved using an Acquity UPLC equipped with BEH RP18 1.7 µm, 2.1 × 100 mm column. Mass spectrometric analysis of the analyte ions was carried out using electrospray with positive ionization and multiple reaction monitoring mode. The assay was linear over a concentration range of 1–100 ng/mL for ketamine and norketamine, and 0.1–10 ng/mL for dehydronorketamine. Acceptable intra-day and inter-day accuracy and precision were observed for all the analytes. High recovery of the analytes and minimal matrix effect were observed. Stability of analytes was confirmed at the tested conditions. This assay was successfully used to measure analytes in human milk samples collected from lactating women enrolled in a clinical research study. This is the first validated method that simultaneously quantified ketamine and its metabolites in human milk.

PCPro: a clinically accessible, circulating lipid biomarker signature for poor-prognosis metastatic prostate cancer

BackgroundUsing comprehensive plasma lipidomic profiling from men with metastatic castration-resistant prostate cancer (mCRPC), we have previously identified a poor-prognostic lipid profile associated with shorter overall survival (OS). In order to translate this biomarker into the clinic, these men must be identifiable via a clinically accessible, regulatory-compliant assay.MethodsA single regulatory-compliant liquid chromatography-mass spectrometry assay of candidate lipids was developed and tested on a mCRPC Discovery cohort of 105 men. Various risk-score Cox regression prognostic models of OS were built using the Discovery cohort. The model with the highest concordance index (PCPro) was chosen for validation and tested on an independent Validation cohort of 183 men.ResultsPCPro, the lipid biomarker, contains Cer(d18:1/18:0), Cer(d18:1/24:0), Cer(d18:1/24:1), triglycerides and total cholesterol. Within the Discovery and Validation cohorts, men who were PCPro positive had significantly shorter OS compared to those who were PCPro negative (Discovery: median OS 12.0 months vs 24.2 months, hazard ratio (HR) 3.75 [95% confidence interval (CI) 2.29–6.15], p < 0.001, Validation: median OS 13.0 months vs 25.7 months, HR = 2.13 [95% CI 1.46–3.12], p < 0.001).ConclusionsWe have developed PCPro, a lipid biomarker assay capable of prospectively identifying men with mCRPC with a poor prognosis. Prospective clinical trials are required to determine if men who are PCPro positive will benefit from therapeutic agents targeting lipid metabolism.

Plasma Biomarkers of Alzheimer's Disease: A Review of Available Assays, Recent Developments, and Implications for Clinical Practice.

Recently, low-sensitive plasma assays have been replaced by new ultra-sensitive assays such as single molecule enzyme-linked immunosorbent assay (Simoa), the Mesoscale Discovery (MSD) platform, and immunoprecipitation-mass spectrometry (IP-MS) with higher accuracy in the determination of plasma biomarkers of Alzheimer's disease (AD). Despite the significant variability, many studies have established in-house cut-off values for the most promising available biomarkers. We first reviewed the most used laboratory methods and assays to measure plasma AD biomarkers. Next, we review studies focused on the diagnostic performance of these biomarkers to identify AD cases, predict cognitive decline in pre-clinical AD cases, and differentiate AD cases from other dementia. We summarized data from studies published until January 2023. A combination of plasma Aβ42/40 ratio, age, and APOE status showed the best accuracy in diagnosing brain amyloidosis with a liquid chromatography-mass spectrometry (LC-MS) assay. Plasma p-tau217 has shown the best accuracy in distinguishing Aβ-PET+ from Aβ-PET-even in cognitively unimpaired individuals. We also summarized the different cut-off values for each biomarker when available. Recently developed assays for plasma biomarkers have undeniable importance in AD research, with improved analytical and diagnostic performance. Some biomarkers have been extensively used in clinical trials and are now clinically available. Nonetheless, several challenges remain to their widespread use in clinical practice.

Preliminary evidence for developing safe and efficient fecal microbiota transplantation as potential treatment for aged related cognitive impairments.

Recent studies have reported that gut microbiota is closely associated with cognitive fuction. Fecal microbiota transplantation (FMT) may be a potential treatment for cognitive impairment, but its efficacy in patients with cognitive impairment is unknown. This study aimed to investigate the safety and efficacy of FMT for cognitive impairment treatment. Five patients aged 54-80 years (three women) were enrolled in this single-arm clinical trial from July 2021 to May 2022. The Montreal Cognitive Assessment-B (MoCA-B), Activities of Daily Living (ADL), and the cognitive section of the Alzheimer's Disease Assessment Scale (ADAS-Cog) were assessed at days 0, 30, 60, 90, and 180. Additionally, stool and serum samples were obtained twice before FMT was administered and six months after the treatment. The structure of fecal microbiota was analyzed by 16S RNA gene sequencing. Serum samples were analyzed for metabolomics and lipopolysaccharide (LPS)-binding proteins by liquid chromatography-mass spectrometry and enzyme-linked immunosorbent assay, respectively. Safety was assessed based on adverse events, vital signs, and laboratory parameters during FMT and the follow-up period. The MoCA, ADL, and ADAS-Cog scores of patients with mild cognitive impairment (patients C and E) after FMT were improved or maintained compared with those before transplantation. However, patients with severe cognitive impairment (patients A, B, and D) had no worsening of cognitive scores. Fecal microbiota analysis showed that FMT changed the structure of gut microbiota. The results of serum metabolomics analysis suggested that there were significant changes in the serum metabolomics of patients after FMT, with 7 up-regulated and 28 down-regulated metabolites. 3b,12a-dihydroxy-5a-cholanoic acid, 25-acetylvulgaroside, deoxycholic acid, 2(R)-hydroxydocosanoic acid, and P-anisic acid increased, while bilirubin and other metabolites decreased. KEFF pathway analysis indicated that the main metabolic pathways were bile secretion and choline metabolism in cancer. No adverse effects were reported throughout the study. In this pilot study, FMT could maintain and improve cognitive function in mild cognitive impairment by changing gut microbiota structure and affecting serum metabolomics. Fecal bacteria capsules were safe. However, further studies are needed to evaluate the safety and efficacy of fecal microbiota transplantation. ClinicalTrials.gov Identifier: CHiCTR2100043548.