Direct analysis in real time mass spectrometry (DART-MS/MS) for rapid urine opioid detection in a clinical setting.

BACKGROUND AND AIMS: Current laboratory methods for opioid detection involve an initial screening with immunoassays which offers efficient but non-specific results and a subsequent liquid chromatography-tandem mass spectrometry (LC-MS/MS) confirmation which offers accurate results but requires extensive sample preparation and turnaround time. Direct Analysis in Real Time (DART) tandem mass spectrometry is evaluated as an alternative approach for accurate opioid detection with efficient sample preparation and turnaround time. MATERIALS AND METHODS: DART-MS/MS was optimized by testing the method with varying temperatures, operation modes, extraction methods, hydrolysis times, and vortex times. The method was evaluated for 12 opioids by testing the analytical measurement range, percent carryover, precision studies, stability, and method-to-method comparison with LC-MS/MS. RESULTS: DART-MS/MS shows high sensitivity and specificity for the detection of 6-acetylmorphine, codeine, hydromorphone, oxymorphone, hydrocodone, naloxone, buprenorphine, norfentanyl, and fentanyl in urine samples. However, its performance was suboptimal for norbuprenorphine, morphine and oxycodone. CONCLUSION: In this proof-of-concept study, DART-MS/MS is evaluated for its rapid quantitative definitive testing of opioids drugs in urine. Further research is needed to expand its application to other areas of drug testing.

Global Metabolomics Using LC-MS for Clinical Applications.

Metabolomics can be used for a multitude of purposes, including monitoring of treatment effects and for increasing the knowledge of the pathophysiology of a wide range of diseases. Global (commonly referred to as "untargeted") metabolomics is hypothesis-generating and provides the opportunity to discover new biomarkers. Being versatile and having a high degree of selectivity and sensitivity, liquid chromatography-mass spectrometry (LC-MS) is the most common technique applied for metabolomics. We here present our global metabolomics LC-electrospray ionization-MS/MS method. The sample preparation procedures for plasma, serum, dried blood spots, urine, and cerebrospinal fluid are simple and nonspecific to reduce the risk of analyte loss. The method is based on reversed-phase chromatography using a diphenyl column. The high-resolution Q Exactive Orbitrap MS with data-dependent acquisition provides MS/MS spectra of a wide range of analytes. Our method covers a large part of the metabolome regarding hydrophobicity and compound class.

Distinguishing Artifactual Fatty Acid Dimers from Fatty Acid Esters of Hydroxy Fatty Acids in Untargeted LC-MS Pipelines.

Untargeted metabolomics is a powerful profiling tool for the discovery of possible biomarkers of disease onset and progression. Analytical pipelines applying liquid chromatography (LC) and mass spectrometry (MS)-based methods are widely used to survey a broad range of metabolites within various metabolic pathways, including organic acids, amino acids, nucleosides, and lipids. Accurate and complete identification of putative metabolites is an ongoing challenge in untargeted metabolomics studies. Highly sensitive instrumentation can result in the detection of adduct and fragment ions that form reproducibly and contain identifiable ions that are difficult to distinguish from metabolic pathway intermediates, which may result in false-positive identification. At concentrations as low as 10 µM, free fatty acids have been found to form homo- and heterodimers in untargeted metabolomics pipelines that resemble the lipid class fatty acid esters of hydroxy fatty acids (FAHFAs), resulting in misidentification. This chapter details a protocol for LC-MS-based untargeted metabolomics using hydrophilic interaction chromatography (HILIC) that specifically aids in distinguishing artifactual fatty acid dimers from endogenous FAHFAs.

LC-MS/MS Quantification of Endocannabinoids in Tissues.

Endocannabinoids (ECBs) are lipid-derived endogenous molecules with important physiological roles such as regulation of energy balance, immunity, or neural development. Quantitation of ECBs helps better understand their physiological role and modulation of biological processes. This chapter presents the simultaneous quantification of 14 ECBs and related molecules in the brain, liver, and muscle, as well as white and brown adipose tissue using liquid chromatography-tandem mass spectrometry (LC-MS/MS). The dynamic range of the method has been tuned to cover the endogenous concentrations of these analytes given the fact that they are endogenously present at different orders of magnitude. Specifically, three groups are established: 0.5-5000 ng/mL for 2-oleoyl- and 2-linoleoylglycerol and arachidonic acid, 0.05-500 ng/mL for 2-arachidonoylglycerol, and 0.0005-0.5 ng/mL for anandamide, palmitoyl-, palmitoleoyl-, stearoyl-, oleoyl-, linoleoyl-, alpha-linolenoyl-, dihomo-gamma-linolenoyl-, docosahexaenoyl-, and pentadecanoylethanolamide.

Application of Proteomics Technology Based on LC-MS Combined with Western Blotting and Co-IP in Antiviral Innate Immunity.

As an interferon-stimulating factor protein, STING plays a role in the response and downstream liaison in antiviral natural immunity. Upon viral invasion, the immediate response of STING protein leads to a series of changes in downstream proteins, which ultimately leads to an antiviral immune response in the form of proinflammatory cytokines and type I interferons, thus triggering an innate immune response, an adaptive immune response in vivo, and long-term protection of the host. In the field of antiviral natural immunity, it is particularly important to rigorously and sequentially probe the dynamic changes in the antiviral natural immunity connector protein STING caused by the entire anti-inflammatory and anti-pathway mechanism and the differences in upstream and downstream proteins. Traditionally, proteomics technology has been validated by detecting proteins in a 2D platform, for which it is difficult to sensitively identify changes in the nature and abundance of target proteins. With the development of mass spectrometry (MS) technology, MS-based proteomics has made important contributions to characterizing the dynamic changes in the natural immune proteome induced by viral infections. MS analytical techniques have several advantages, such as high throughput, rapidity, sensitivity, accuracy, and automation. The most common techniques for detecting complex proteomes are liquid chromatography (LC) and mass spectrometry (MS). LC-MS (Liquid Chromatography-Mass Spectrometry), which combines the physical separation capability of LC and the mass analysis capability of MS, is a powerful technique mainly used for analyzing the proteome of cells, tissues, and body fluids. To explore the combination of traditional proteomics techniques such as Western blotting, Co-IP (co-Immunoprecipitation), and the latest LC-MS methods to probe the anti-inflammatory pathway and the differential changes in upstream and downstream proteins induced by the antiviral natural immune junction protein STING.

Identifying an Abnormal Phosphorylated Adaptor by Viral Kinase Using Mass Spectrometry.

Mass spectrometers are widely used to identify protein phosphorylation sites. The process usually involves selective isolation of phosphoproteins and subsequent fragmentation to identify both the peptide sequence and phosphorylation site. Immunoprecipitation could capture and purify the protein of interest, greatly reducing sample complexity before submitting it for mass spectrometry analysis. This chapter describes a method to identify an abnormal phosphorylated site of the adaptor protein by a viral kinase through immunoprecipitation followed by LC-MS/MS.

Determination of α-methylenecyclopropylglycine in Shahi and China litchi cultivars at three different maturity stages: A quantitative study using liquid chromatography tandem mass spectrometry.

This study presents the contents of α-methylenecyclopropylglycine, a potentially toxic amino acid, in the peel, pulp and seed fractions of two well-known litchi varieties, namely Shahi and China, over a span of three harvest-seasons. For analysing α-methylenecyclopropylglycine, an LC-MS/MS-based method was validated. The method-accuracies fell within 75-110 % (RSD, <15 %) at 0.1 mg/kg (LOQ) and higher levels. A comparative evaluation of the results in peel, pulp and seed at 30 days before harvest (DBH), 15-DBH, and edible-ripe stage revealed that α-methylenecyclopropylglycine content increased as the litchi seeds grew towards maturity, regardless of the cultivar. In arils, at maturity, the concentration of α-methylenecyclopropylglycine ranged from not-detected to 11.7 µg/g dry weight. The Shahi cultivar showed slightly higher α-methylenecyclopropylglycine content in comparison to China litchi. This paper presents the first known analysis of combined seasonal data on different fruit components at various growth stages for the two chosen litchi cultivars grown in India.

Evidence to support cultivated fruiting body of Ophiocordyceps sinensis (Ascomycota)'s role in relaxing airway smooth muscle.

ETHNOPHARMACOLOGICAL RELEVANCE: Ophiocordyceps sinensis (O. sinensis) is a genus of Ascomycete fungus that is endemic to the alpine meadows of the Tibetan Plateau and adjoining Himalayas. It has been used traditionally as a tonic to improve respiratory health in ancient China as well as to promote vitality and longevity. Bioactive components found in O. sinensis such as adenosine, cordycepin, 3-deoxyadenosine, L-arginine and polysaccharides have gained increasing interest in recent years due to their antioxidative and other properties, which include anti-asthmatic, antiviral, immunomodulation and improvement of general health. AIM OF THE STUDY: This study's primary aim was to investigate the effect of a cultivated fruiting body of O. sinensis strain (OCS02®) on airways patency and the secondary focus was to investigate its effect on the lifespan of Caenorhabditis elegans. MATERIALS AND METHODS: A cultivated strain, OCS02®, was employed and the metabolic profile of its cold-water extract (CWE) was analysed through liquid chromatography-mass spectrometry (LC-MS). Organ bath approach was used to investigate the pharmacological properties of OCS02® CWE when applied on airway tissues obtained from adult male Sprague-Dawley rats. The airway relaxation mechanisms of OCS02® CWE were explored using pharmacological tools, where the key regulators in airway relaxation and constriction were investigated. For the longevity study, age-synchronised, pos-1 RNAi-treated wild-type type Caenorhabditis elegans at the L4 stage were utilised for a lifespan assay. RESULTS: Various glycopeptides and amino acids, particularly a high concentration of L-arginine, were identified from the LC-MS analysis. In airway tissues, OCS02® CWE induced a significantly greater concentration-dependent relaxation when compared to salbutamol. The relaxation response was significantly attenuated in the presence of NG-Nitro-L-arginine methyl ester (L-NAME), 1H-[1,2,4]oxadiazolo [4,3-a]quinoxalin-1-one (ODQ) and several K+ channel blockers. The longevity effect induced by OCS02® CWE (5 mg/mL and above) was observed in C. elegans by at least 17%. CONCLUSIONS: These findings suggest that the airway relaxation mechanisms of OCS02® CWE involved cGMP-dependent and cGMP-independent nitric oxide signalling pathways. This study provides evidence that the cultivated strain of OCS02® exhibits airway relaxation effects which supports the traditional use of its wild O. sinensis in strengthening respiratory health.

LC-MS coupled with diagnostic ion strategy facilitated the discovery of 5-methylcoumarin meroterpenoids from Gerbera piloselloides.

Plant-derived natural products remain crucial in drug development. However, the identification of undescribed natural products is becoming increasingly challenging. A comprehensive strategy combining LC-MS with diagnostic ions was proposed for the discovery of undescribed 5-methylcoumarin meroterpenoids. Thirteen undescribed 5-methylcoumarin meroterpenoids, including five pairs of enantiomers (1a/1b and 5a/5b-8a/8b), were isolated from the whole plant of Gerbera piloselloides. Their structures and absolute configurations were unambiguously determined based on their spectroscopic data, calculated and experimental ECD data and X-ray diffraction analysis. Bioassays conducted on scopolamine-induced injury PC12 cells revealed that compounds 5a/5b, 7a/7b and 8a/8b possessed mild protective effects. Additionally, compounds 2 and 8 showed notable IL-6 inhibition in lipopolysaccharide-induced BEAS-2B cells.

Generating and characterizing a comprehensive panel of CHO cells glycosylation mutants for advancing glycobiology and biotechnology research.

This report describes the development and characterization of a comprehensive collection of CHO cell glycosylation mutants with significant potential for advancing glycobiology and biotechnology. EPO-Fc and trastuzumab, two model molecules, were produced using these mutants to assess the effects of mutated glycogenes, and LC-MS/MS analysis was employed to quantitatively analyse their N-glycans. EPO-Fc exhibited exclusively homogeneous Man9 glycans only when nearly all α-mannosidases in the genome were inactivated, except lysosomal MAN2B1. Some mutants lacking GnT-I activity produce mostly Man5 N-glycans, while their O-glycan and glycolipid profiles can differ due to other mutations in the cell. GnT-II deficiency prevents GnT-V from adding GlcNAc to the core N-glycan, resulting in branches attaching solely to the α1,3-linked mannose, leaving the α1,6-linked mannose free. The mutant-produced antibody's single-branched glycan contains more sialic acid than the dual-branched glycans produced in CHO-K1 cells. Trastuzumab produced in these mutants provided insights into how Fc N-glycans impact the antibody's interaction with FcγR1 and FcγR2a, FcγR3a, and their influence on antibody-dependent cellular cytotoxicity (ADCC). In the study of Fc glycans in Fc-FcγR1 and FcγR2a interactions, we observed a consistent glycan-related impact on binding to both receptors, indicating a common interaction mechanism between Fc glycans and both FcγRI and FcγRIIa. CHO mutants produced trimeric gp120 demonstrated distinct reactivity with multiple broadly neutralizing anti-HIV antibodies, confirming the involvement of gp120 glycans in interactions with specific broadly neutralizing antibodies. Finally, one of the mutants produced human ß-glucocerebrosidase with uniform Man5 N-glycans, showcasing its potential for glycoengineered production and enhancement in therapeutic efficacy.

A validated LC-MS/MS method for the simultaneous quantification of iothalamate and hippuran in serum and urine for non-radioactive kidney function assessment.

A novel liquid chromatography-tandem mass spectrometry method is described for the quantitative determination of the kidney function markers iothalamate and hippuran in human serum and urine. It is based on protein precipitation with methanol followed by dilution of the supernatant for serum and simple dilution for urine. The polar analytes are chromatographically separated by a 6.5-min gradient on a low-ligand density reversed-phase column; detection is performed by electrospray ionization tandem mass spectrometry in the positive ion mode against stable-isotope labeled internal standards. The results of a thorough method validation show that iothalamate and hippuran can be simultaneously quantified in the concentration ranges 0.500-30.0 ng/mL and 10.0-5000 ng/mL for serum and urine, respectively, with values for CV and absolute bias not exceeding 10 %, and with sufficient stability in all relevant matrices and solvents. The method was successfully applied for the analysis of serum and urine samples of multiple individuals who received both iothalamate and hippuran.

Automated kapok fiber-based pipette-tip solid-phase extraction coupled with liquid chromatography-tandem mass spectrometry for rapid and sensitive analysis of tyrosine kinase inhibitors in plasma.

This study delineates the development of a novel automated pipette-tip solid-phase extraction (SPE) methodology, employing kapok fiber as a naturally efficient and cost-effective adsorbent for the selective extraction of eleven tyrosine kinase inhibitors (TKIs) from plasma. The uniqueness of this method lies in its assembly, where kapok fibers are ingeniously wrapped around a stainless-steel spring within the pipette tip, ensuring an obstruction-free central space for effortless solution aspiration and dispensation. This design significantly minimizes backpressure, enhancing operational efficiency and ensuring compatibility with pipettors, including the implementation of an electric pipettor to streamline the sample preparation process and facilitate automation. The method's analytical performance, rigorously validated through liquid chromatography-tandem mass spectrometry, exhibits outstanding linearity in ranges of 0.1/0.5-200 ng mL-1 (R² > 0.993), commendable accuracy (86.3%-114.8%), and consistent precision (3.4-11.3%), alongside remarkably low detection limits that span from 0.024 to 0.130 ng mL-1. The assembly of kapok fiber within the pipette tip, in this unique configuration, results in a practical, cost-effective, eco-friendly, and automated pipette-tip SPE method. This innovation signifies a significant advancement in bioanalytical methodologies, offering an efficient and sustainable approach for extracting analytes from complex biological samples. This process notably enhances both the sensitivity and selectivity of subsequent instrumental analyses.

In vivo hypotensive effect of a chemically characterised extract from the leaves of Lippia alba (Mill.) N.E.Br.

Lippia alba (erva-cidreira) is often mentioned in Brazilian ethnopharmacological studies. Although its leaves have been used to treat hypertension, few studies have evaluated its hypotensive effects. This work aimed to evaluate the haemodynamic effects of Lippia alba methanolic extract and to characterise its chemical composition. Normotensive rats received an intravenous injection of L. alba extract. Systolic, diastolic, mean arterial pressures, and electrocardiographic data were analysed.1H-qNMR and LC-MS were used to assess the chemical composition. L. alba extract had significant hypotensive effects on systolic, diastolic, and mean arterial pressure. Acteoside was identified as major compound (292.6 ± 2.7 mg/g). Sixty-one other compounds were tentatively identified, mainly phenylethanoids, flavonoids, and iridoids. L. alba extract reduces systolic, diastolic, mean arterial pressure, and appears to be associated with a reduction in heart rate. Acteoside, a known hypotensive compound, may be responsible for these effects, but other structurally similar minority compounds may also contribute.

Clinical implications of opioid parent-metabolite ratios.

BACKGROUND: The opioid epidemic has underscored the importance of urine drug testing in the management of chronic pain. However, interpreting test results can be challenging, especially in scenarios where medications may have been directly added to urine samples to simulate compliance. METHODS: We conducted a retrospective analysis of 9,690 opioid testing results using liquid chromatography-tandem mass spectrometry (LC-MS/MS). The study aimed to define the expected ratios between parent drugs and metabolites for eight commonly prescribed opioids. Cases with a parent-metabolite ratio above the 95th percentile were subjected to chart review. RESULTS: A total of 13 cases appeared likely consistent with simulated compliance with buprenorphine, 2 with methadone, 14 with oxycodone, and one with hydrocodone. The unusual patterns of parent-metabolite ratio can also be associated with hyperacute drug exposures/use, pharmaceutical impurity, or underlying liver enzyme deficiency. Furthermore, patients who failed the decision limits could exhibit other illicit use or aberrant behaviors. CONCLUSION: Laboratories conducting LC-MS/MS-based opioid testing can more objectively identify anomalies by analyzing parent-metabolite ratios. When in consultation with providers, laboratories can point to these data when suggesting the possibility of simulated compliance and help identify cases warranting further investigation.

Development of a Versatile High-through-put Oligonucleotide LC-MS Method to Accelerate Drug Discovery.

Liquid chromatography-mass spectrometry (LC-MS) is an effective tool for high-throughput quantification of oligonucleotides that is crucial for understanding their biological roles and developing diagnostic tests. This paper presents a high-throughput LC-MS/MS method that may be versatilely applied for a wide range of oligonucleotides, making it a valuable tool for rapid screening and discovery. The method is demonstrated using an in-house synthesized MALAT-1 Antisense oligonucleotide (ASO) as a test case. Biological samples were purified using a reversed liquid-liquid extraction process automated by a liquid handling workstation and analyzed with ion-pairing LC-MS/MS. The assay was evaluated for sensitivity (LLOQ = 2 nM), specificity, precision, accuracy, recovery, matrix effect, and stability in rat cerebrospinal fluid (CSF) and plasma. Besides some existing considerations such as column selection, ion-pairing reagent, and sample purification, our work focused on the following four subtopics: 1) selecting the appropriate Multiple Reaction Monitoring (MRM) transition to maximize sensitivity for trace-level ASO in biological samples; 2) utilizing a generic risk-free internal standard (tenofovir) to avoid crosstalk interference from the oligo internal standard commonly utilized in the LC-MS assay; 3) automating the sample preparation process to increase precision and throughput; and 4) comparing liquid-liquid extraction (LLE) and solid-phase extraction (SPE) as sample purification methods in oligo method development. The study quantified the concentration of MALAT-1 ASO in rat CSF and plasma after intrathecal injection and used the difference between the two matrices to evaluate the injection technique. The results provide a solid foundation for further internal oligonucleotide discovery and development.

Comprehensive chromatographic analysis of Belumosudil and its degradation products: Development, validation, and In silico toxicity assessment.

Following ICH guidelines, the stability of Belumosudil, a novel protein kinase inhibitor, was tested under different stress conditions (hydrolytic, oxidative, photolytic, and thermal). A selective and efficient separation of Belumosudil and its degradation products was achieved using a Quality by Design approach. In-silico predictions using Zeneth Nexus® software were employed to assess the compound's degradation under various stress scenarios. The methodology developed through experimental design analyzed crucial process parameters connected with chromatographic systems. Reversed-phase high-performance liquid chromatography with a C18 column and a gradient mobile phase of acetonitrile and 25 mM ammonium hydrogen carbonate buffer (pH 5.6) were utilized. For structural characterization and identification of degradation products, UPLC-quadrupole tandem mass spectrometry was employed. Four distinct degradation products were identified under different stress settings. The method was thoroughly validated, assessing accuracy, selectivity, repeatability, system suitability, and linearity range (5.0-120.0 µg/mL). To predict mutagenicity and toxicity, DEREK Nexus® software was used. Two degradation products were predicted to induce skin sensitization, irritation, and hepatotoxicity in humans.

Assessment of the in vitro metabolic stability of CEP-37440, a selective FAK/ALK inhibitor, in HLMs using fast UPLC-MS/MS method: in silico metabolic lability and DEREK alerts screening.

Introduction: CEP-37440 was synthesized and supplied by the research and development division of Teva Branded Pharmaceutical Products (West Chester, PA, United States). CEP-37440 represents a newly developed compound that exhibits selectivity inhibition of Focal Adhesion Kinase and Anaplastic Lymphoma Kinase FAK/ALK receptors, demonstrating novel characteristics as an orally active inhibitor. The simultaneous inhibition of ALK and FAK can effectively address resistance and enhance the therapeutic efficacy against tumors through a synergistic mechanism. Methods: The objective of this research was to create an LC-MS/MS method that is precise, efficient, environmentally friendly, and possesses a high level of sensitivity for the quantification of CEP-37440 in human liver microsomes (HLMs). The aforementioned approach was subsequently employed to evaluate the metabolic stability of CEP-37440 in HLMs in an in vitro setting. The validation procedures for the LC-MS/MS analytical method in the HLMs were performed following the bio-analytical method validation guidelines set out by the US-FDA. The AGREE program was utilized to assess the ecological impacts of the current LC-MS/MS methodology. Results and Discussion: The calibration curve linearity was seen in the range of 1-3000 ng/mL. The inter-day accuracy (% RE) exhibited a range of -2.33% to 3.22%, whilst the intra-day accuracy demonstrated a range of -4.33% to 1.39%. The inter-day precision (% RSD) exhibited a range of 0.38% to 3.60%, whilst the intra-day precision demonstrated a range of 0.16% to 6.28%. The determination of the in vitro half-life (t1/2) and moderate intrinsic clearance (Clint) of CEP-37440 yielded values of 23.24 min and 34.74 mL/min/kg, respectively. The current manuscript is considered the first analytical study for CEP-37440 quantification with the application to metabolic stability assessment. These results suggest that CEP-37440 can be categorized as a pharmaceutical agent with a moderate extraction ratio. Consequently, it is postulated that the administration of CEP-37440 to patients may not lead to the accrual of dosages within the human organs. According to in silico P450 metabolic and DEREK software, minor structural alterations to the ethanolamine moiety or substitution of the group in drug design have the potential to enhance the metabolic stability and safety profile of novel derivatives in comparison to CEP-37440.

Personalization of pharmacotherapy with sirolimus based on volumetric absorptive microsampling (VAMS) in pediatric renal transplant recipients-from LC-MS/MS method validation to clinical application.

BACKGROUND: The benefits of pharmacotherapy with sirolimus (SIR) in pediatric transplant recipients are well established. Traditionally, whole blood samples have been used to measure SIR concentrations. Volumetric Absorptive Microsampling (VAMS) is an alternative sampling strategy suitable for Therapeutic Drug Monitoring (TDM). In this study, we developed and validated two liquid chromatography-tandem mass spectrometry (LC-MS/MS) methods for determining SIR concentrations in whole blood (WB) and capillary whole blood samples collected using a VAMS-Mitra™ device. METHODS: We used protein precipitation during WB sample preparation and dispersive liquid-liquid microextraction (DLLME) with methyl tert-butyl ether for VAMS sample preparation to optimise the analyte extraction process. The described validation protocols were cross-validated, confirming the equivalence of the whole-blood and VAMS-based methods. Furthermore, the developed methods were evaluated in two three-level rounds of an external proficiency-testing scheme. RESULTS: The analytical methods were successfully validated within the calibration range of SIR (0.5-60 ng/ml). The validation parameters met the European Medicines Agency (EMA) and the International Association of Therapeutic Drug Monitoring and Clinical Toxicology (IATDM&CT) acceptance criteria. No hematocrit (tested in the range of 24.3-64.1%), matrix, or carry-over effects were observed. Cross-validation confirmed the interchangeability between VAMS-LC-MS/MS and WB-LC-MS/MS methods. The developed methods were successfully implemented for SIR determination in 140 clinical samples (70 each of WB and VAMS) from pediatric renal transplant recipients, demonstrating their practicality and reliability. CONCLUSION: The VAMS-based method has been rigorously tested and is clinically equivalent to the reference WB-LC-MS/MS method. Additionally, clinical validation confirmed the utility of the presented methods for TDM of the SIR in the pediatric population after renal transplantation.

Elucidating the effective age for dietary restriction and the key metabolites involved.

Dietary restriction (DR) extends lifespan in various species, but its effect at different ages, especially when started later, is unclear. This study used Caenorhabditis elegans to explore the impact of DR at different ages. Worms were divided into control and DR groups, with daily survival monitored. To confirm the occurrence of DR, the expression of DR-sensitive genes namely acdh-1, pyk-1, pck-2 and cts-1 were determined using RT-qPCR. Liquid chromatography mass spectrometry (LC-MS) was employed to observe the changes in metabolites affected by DR. The results indicated that young worms subjected to mild DR displayed the longest lifespan, highlighting the effectiveness of initiating DR at a young age. Increased expression of acdh-1 and pck-2 suggests activation of beta-oxidation and gluconeogenesis, while decreased cts-1 expression indicates a reduced citric acid cycle, further supporting the observed effects of DR in these worms. Metabolomic results indicated that DR decreased the activity of mechanistic Target of Rapamycin (mTOR) and the synthesis of amino acids namely leucine, tyrosine and tryptophan to conserve energy for cell repair and survival. DR also decreased levels of N-acetyl-L-methionine and S-adenosyl-methionine (SAM) in methionine metabolism, thereby promoting autophagy, reducing inflammation, and facilitating the removal of damaged cells and proteins. In conclusion, initiating dietary restriction early in life extends the lifespan by modulating amino acid metabolism and enhancing the autophagy pathway, thereby maintaining cellular wellbeing.

Evaluation of 4 quantification methods for monitoring 16 antidepressant drugs and their metabolites in human plasma by LC-MS/MS.

BACKGROUND: Therapeutic drug monitoring for antidepressants (ADs) is vital due to the potentially serious consequences and disputes related to medical events. Therefore, we created a quick and convenient analysis way for separation and quantification of ADs. METHODS: To ensure quantitative stability, we divided the 16 ADs or their metabolites into 4 pools (AD1-AD4), considering the hospital frequency that the clinician prescribed, the physicochemical properties of medicines, and the calibration range of selected ADs. After precipitation with methanol, the analytes were eluted for at least 3.5 min on a BEH C18 analytical column by different gradient elution methods. RESULTS: The LLOQ and LOD were 1.25-10 ng/mL and 0.42-5 ng/mL, respectively. High precision (<12 %) and accuracy (87.07-111.47 %) were demonstrated by quality control samples both within and between days. All the compounds were stable at room temperature and within -80 °C. CONCLUSION: The method is of wide clinical and laboratory interest due to simpler sample cleanup, shorter chromatographic run times, and wider calibration range compared to other methods.