Liquid Chromatography-tandem Mass Spectrometry Method Research Articles

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

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

A rapid liquid chromatography-tandem mass spectrometry based method for the detection of Tet(X) resistance gene in Enterobacteriaceae

There is a major public health threat posed by antibiotic resistance around the world. Tigecycline overcomes the resistance mechanisms of traditional tetracyclines and is often seen as the final resort in combating infections caused by bacteria resistant to multiple drugs. However, the introduction of new mobile tet(X) tetracycline destructases is leading to a notable rise in tigecycline resistance. Therefore, a rapid detection method is needed to monitor the spread of tigecycline resistance. In this study, a novel liquid chromatography-tandem mass spectrometry (LC-MS/MS) method to detect tet(X) in bacterial isolates was developed. This method utilized the analysis by LC-MS/MS of metabolite ratios to determine the presence of tet(X). Bacterial suspensions were co-incubated with tigecycline for 1 h, where tet(X) destructase inactivated tigecycline, making a particular metabolite with a 16-Da change in mass. The characterized quantitative ion pairing of tigecycline in the ESI positive mode was observed at 586.1 → 569.1 m/z. The oxygenated tigecycline detection was established at 602.2 → 529.1 m/z. A model was established using 35 tet(X)-positive and 15 tet(X)-negative Enterobacteriaceae strains in this study to optimize the cutoff value. Applying the model to analyze 70 bacterial isolates, the sensitivity of the LC-MS/MS test was 98.9% compared to polymerase chain reaction (PCR), and specificity was 100%. This method is rapid and easy to operate, providing results within 1 h, making it more suitable for routine use in clinical microbiology laboratories.

Measuring Lp(a) particles with a novel isoform-insensitive immunoassay illustrates efficacy of muvalaplin.

Lipoprotein(a) [Lp(a)] is a cardiovascular risk factor, and there is considerable interest in developing Lp(a)-lowering therapeutics for cardiovascular prevention. Current commercial Lp(a) assays measure total apolipoprotein(a) [apo(a)] and may be insufficient to accurately measure Lp(a) concentrations and determine Lp(a) lowering by a new class of small-molecule Lp(a) formation inhibitors such as muvalaplin. We developed a novel immunoassay that measures only Lp(a) particles. This intact Lp(a) assay demonstrated robust analytical performance, was insensitive to apo(a) isoform size, and correlated with a liquid chromatography-tandem mass spectrometry method. Muvalaplin phase I multiple ascending dose study samples and lepodisiran, a small interfering RNA that lowers Lp(a), phase I single ascending dose study samples were analyzed using the intact Lp(a) assay and commercial assays. The Lp(a)-lowering efficacy of muvalaplin was underestimated by the commercial assay measuring total apo(a) compared with the intact Lp(a) assay specifically measuring Lp(a) particles. In contrast, the Lp(a)-lowering effect of lepodisiran was clinically comparable between the intact Lp(a) assay and commercial assay. This novel intact Lp(a) assay provides a more accurate approach for the assessment of Lp(a)-lowering agents and study of Lp(a)-associated risk compared with currently available assays.

Application of Advanced High-Resolution Mass Spectrometric Techniques for the Analysis of Losartan Potassium Drug Substance Degradation Products: From Nontargeted to Targeted Screening.

Advances in techniques for quality analysis allow for a more detailed examination of drug impurities. High-resolution mass spectrometry (HRMS) contributes to detecting both known and unknown impurities. In this study, a combination of a nontargeted and targeted screening approach was established and applied to the detailed degradation profile of the losartan potassium (LP) drug substance. Through general unknown comparative screening (GUCS), 35 degradation products (DPs) were detected; of these, 10 DPs were confirmed with reference substances. DP-1, DP-2, DP-3, and DP-6 were the first characterized, as per previous studies; the other twenty-four were newly identified. In addition, a liquid chromatography-tandem mass spectrometry method was developed for the determination of the ten DPs. It was sensitive, with the limit of quantitation of analytes ranging from 0.01 to 0.5ng mL-1. Two newly characterized DPs, DP-1 and DP-2, were determined in active pharmaceutical ingredient solutions. This study introduced a new approach using broad screening to analyze degradation impurity profiles in LP drug substance, aiding in the identification of low-level impurities or those without reference substances. Additionally, the sensitive determination method developed allows for the precise quantification and control of ten DPs at trace levels in LP drug substances or products.

Development and Validation of a Sensitive LC-MS/MS Method for Determination of Lenvatinib and Its Major Metabolites in Human Plasma and Its Application in Hepatocellular Carcinoma Patients.

Lenvatinib has been demonstrated effective in advanced hepatocellular carcinoma (HCC), but the pharmacokinetic-pharmacodynamics behavior of lenvatinib and its metabolites remains unclear. To investigate the pharmacokinetic-pharmacodynamics behavior of lenvatinib and its active metabolites in advanced HCC patients, it is important to develop a simple and rapid method to analyze the exposures of lenvatinib and its metabolites in human samples. Here, we established and validated a simple and rapid method for determining lenvatinib and its three major metabolites, descyclopropyl lenvatinib (M1), O-demethyl lenvatinib hydrochloride (M2), and lenvatinib N-Oxide (M3) by liquid chromatography-tandem mass spectrometry method. Lenvatinib and its main metabolites were separated on an X-Terra RP18 column (50 × 2.1mm, 3.5 µm) at 35°C within 3min, and the analytes were isocratically eluted with the mobile phase of methanol-water (10:90, v/v) containing 0.1% of formic acid at a flow rate of 0.15mL/min. The calibration range was 1-1000ng/mL for lenvatinib, while 0.1-100ng/mL for M1-M3 under positive electrospray ionization mode. The inter- and intra-batch precisions and accuracy were acceptable for lenvatinib and its metabolites. This method was successfully applied to measure lenvatinib and its metabolites in plasma samples from HCC patients, which provides a robust tool for pharmacokinetic-pharmacodynamics studies of lenvatinib.

Preparation and evaluation of Puerarin-loaded PLGA nanoparticles for improving oral bioavailability in SD rats

BackgroundPuerarin (Pue) is an isoflavone compound with significant therapeutic effect on cardiovascular diseases, but its poor water solubility and low oral bioavailability limit clinical application. MethodsIn this study, Pue was prepared into PLGA nanoparticles (Pue-PLGA NPs) by emulsion solvent volatilization method. The morphology, particle size, Zeta potential, X-ray diffraction (XRD), and fourier transform infrared (FTIR) of the NPs were characterized. Additionally, their stability and in vitro release were evaluated. SD rats were orally administered wtih Pue and Pue-PLGA NPs, and a high performance liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was established to determine the concentration of blood samples and to investigate the pharmacokinetic behaviour of Pue and Pue-PLGA NPs in rats. ResultsThe NPs were observed by transmission electron microscopy (TEM) as regular spheroids and uniformly dispersed. The average particle size of the NPs was (167.1±5.26）nm, the Zeta potential was (-29.88±2.46)mV, the encapsulation rate was (83.12 %±4.73 %) and the drug loading capacity was (7.75 %±1.81 %). The results of in vitro release showed that the drug was released slowly and continuously from the NPs, reaching the release platform in 24 h, and the cumulative release amount was (88.55±2.86) %. The pharmacokinetic results showed that the AUC0–24, AUC0-∞, Cmax, Tmax, t1/2, MRT0–24 and MRT0-∞ of Pue-PLGA NPs were 2.196, 1.978, 1.327, 1.5, 1.385, 3.915 and 3.140 times of Pue, respectively. The relative bioavailability was (197.82±25.28) %. ConclusionThese results indicated that the prepared nanoparticles had small particle size, high encapsulation rate, drug loading capacity and good slow-release effect, and could significantly improve the oral bioavailability of Pue in rats.

Pharmacokinetics and Bioequivalence Evaluation of Trazodone Hydrochloride Sustained-Release Tablets in Healthy Chinese Volunteers.

The aim of this study was to investigate the pharmacokinetics, bioequivalence, and safety of generic trazodone hydrochloride sustained-release tablet and its reference listed product in healthy Chinese subjects. An open, randomized, single-dose, and 2-period crossover study was involved under fasting and fed conditions, with a 7-day washout period. A single oral dose of 150mg of 2 trazodone hydrochloride sustained-release tablets was administered to 84 healthy volunteers, with 36 in the fasting group and 48 consuming a high-fat diet, respectively. The plasma concentrations of trazodone were analyzed using a liquid chromatography-tandem mass spectrometry method, and pharmacokinetic parameters were obtained from concentration-time profiles. The geometric mean ratio with 90% confidence intervals of the maximum trazodone concentration, area under the plasma concentration-time curve (AUC) from time 0 to the last measurable concentration, and AUC from time 0 to infinity were within the bioequivalence acceptance criteria (80%-125%) under fasting and fed conditions, which indicated that the test and reference formulations were bioequivalent. Compared with the fasting study, the concomitant administration of trazodone with a high-fat diet had a negligible influence on the drug pharmacokinetic behavior. Adverse events were recorded, and no serious adverse events were observed during either fasting or fed conditions. Trazodone has proven to have an acceptable safety profile in the Chinese population, with bioequivalence successfully established under both fasting and fed conditions.

Fecal fatty acid-linked bile acid profiles in pediatric patients with ulcerative colitis

ObjectiveEffect of gut dysbiosis on fatty acid (FA) linked 3β-hydroxy-bile acid esters (FA-isoBAs) formation is currently unknown. This study aimed to investigate the profile of FA-isoBAs in fecal samples from pediatric patients with ulcerative colitis (UC). MethodsFecal samples were collected from seven pediatric patients diagnosed with UC and seven age-matched healthy controls. Liquid chromatography-tandem mass spectrometry (LC/MS) method was set up for quantifications of thirteen different FA-isoBAs, including three new FA-isoBAs which had never been characterized. Method validation tests were performed with optimization of sample preparation. Statistical analyses were conducted to compare FA-isoBA concentrations between UC patients and healthy controls. ResultsThe LC/MS method had sufficient linearity (r > 0.998), with the low detection limit (0.03–2.82 nmol/g stool), and limits of quantification (0.10–9.40 nmol/g stool) for all FA-isoBAs. The total FA-isoBAs concentration in UC patients was significantly lower than healthy controls, regardless of the degree of the disease severity. The UC subjects had markedly increased primary bile acid (BA) levels with decreased secondary BAs. Propionic acid-linked isoBA and stearic acid-linked 12-oxo-isolithocholic acid were newly identified from healthy subjects but they were not present in UC subjects. In agreement with the previous report, composition of long-chain (C16-C18) FA-linked BAs dominated over short-chain FA-linked BAs both in healthy and disease subjects. ConclusionThe present study reported the fecal FA-isoBA profiles in pediatric UC patients for the first time. The results open the door to the new field that investigates the role of these microbial-driven BAs in gastrointestinal health.

Application of magnetic bead-assisted liquid chromatography-tandem mass spectrometry method for analyzing serum bile acid profiles: Advancing toward automated mass spectrometry

BackgroundMagnetic bead-assisted extraction technology offers potential for automating mass spectrometry processes, yet research in this area remains limited. Here, we developed and validated a magnetic bead extraction method to simultaneously analyze 13 bile acid profiles in human serum. MethodsA magnetic bead-assisted liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was established for serum bile acid profiles. The linearity, accuracy, precision, matrix effect, carryover, stability test and interference test of the LC-MS/MS method were assessed. Comparative analysis involving 20 pregnant women evaluated different methodologies for diagnosing and monitoring intrahepatic cholestasis of pregnancy (ICP). ResultsThe magnetic bead extraction method demonstrated analytical performance comparable to the traditional protein precipitation method. The method exhibited linear responses across the clinical reference interval for all 13 bile acids. Meanwhile, the recovery rates for bile acid analytes ranged from 85.14 to 114.43%, and the repeatability was within 1.68 to 10.83%, meeting acceptance criteria. Our magnetic bead-assisted extraction method showed no obvious matrix effects and carryover effects. The extracted specimens remained stable in the autosampler for 24 h, and the interference test suggested the bile acid profile results would not be significantly affected under different interfering substances. Furthermore, comparative studies with traditional protein precipitation method showed comparable results, and our method exhibited robust clinical consistency in monitoring the efficacy of ICP patients. ConclusionsWe developed a straightforward magnetic bead extraction method coupled with LC-MS/MS for bile acid profiles. It can be applied to the clinical routine diagnosis of ICP and facilitate the automated mass spectrometry processes.

A Validated Liquid Chromatography-Tandem Mass Spectrometry Method for the Determination of Methylophiopogonanone A in Rat Plasma and Its Application to Pharmacokinetic Study.

Methylophiopogonanone A (MOA) is one of the homoisoflavonoids isolated from Ophiopogon japonicus, which has been demonstrated to have extensive pharmacological activities. The aim of this study was to develop and validate a liquid chromatography-tandem mass spectrometry method for the measurement of MOA in rat plasma. Methylophiopogonanone B (MOB) was used as internal standard. After precipitation with acetonitrile, the samples were separated using a Waters ACQUITY HSS T3 column with 0.1% formic acid solution and acetonitrile as mobile phase. Mass detection was monitored using multiple reactions monitoring mode with precursor-to-product ion transition of m/z 343.2 > 135.1 for MOA and m/z 329.2 > 121.1 for internal standard. The assay showed good linearity over the concentration range of 1-1000 ng/mL, with correlation coefficient > 0.997. The lower limit of quantification (LLOQ) was 1 ng/mL. Acetonitrile-mediated precipitation showed high extraction efficiency (> 80%). The accuracy and precision were within the acceptable limits. MOA was demonstrated to be stable in rat plasma under the tested storage conditions. After validation, the proposed method was applied to the pharmacokinetic study of MOA in rat plasma, and the data revealed that MOA showed rapid absorption and elimination from rat plasma. The oral bioavailability of MOA in rat was 24.5%.

Determination of Hormonal Growth Promotants in Beef Using Liquid Chromatography-Tandem Mass Spectrometry.

Hormonal growth promotants (HGPs) are a class of pharmaceutical agents commonly administered to cattle in the United States to improve growth rates of the animal, alter behavior, or to improve the desired characteristics of retail cuts of meat. There is a concern that low residual concentrations of HGPs may remain in tissue after slaughter, and consumption of tissues containing these compounds may increase the risk of adverse health outcomes, including cancer. Sensitive and selective methods are necessary to assess exposure of HGPs by populations that consume meat products from animals that may have been administered HGPs. A liquid chromatography-tandem mass spectrometry method was developed and validated to detect the low-level presence of HGPs including estradiol, testosterone, estradiol benzoate, melengestrol, melengestrol acetate, progesterone, testosterone propionate, trenbolone, trenbolone acetate, and α-zearalanol in retail cuts of meat following a liquid-liquid extraction using a high pH solution with 30-50× less mass of meat required as compared to similar approaches. Good chromatographic performance and sensitivity was achieved utilizing ammonium fluoride as a mobile phase additive without the need for derivatization. Validation parameters including accuracy, precision, recovery, matrix effects, limits of detection, limits of quantitation, linear range, and stability were determined. The limits of detection ranged from 0.1 to 1.0 ng/g, depending on the compound, with adequate accuracy and precision without the need for extensive sample preparation approaches.

Development and Clinical Validation of Liquid Chromatography-Tandem Mass Spectrometry for Measuring Ruxolitinib in Steroid-Refractory Graft-Versus-Host Disease: A First Step Towards Optimized Treatment.

This non-interventional, prospective, single-center study aimed to develop a technique to measure ruxolitinib (RUX) concentrations and provide preliminary data on the distribution of plasma drug levels in patients with steroid refractory (SR) GvHD. Between April 2023 and May 2024, we analyzed 48 blood samples from 29 patients with SR-GvHD. Median individual plasma concentrations varied across different RUX doses and largely overlapped: 39.2 ng/mL at 10 mg b.i.d (range: 0-73), 13.1 ng/mL at 10-5 mg (range, 6.1-35.6), and 31.6 ng/mL at 5 mg b.i.d (range: 0.7-99.9). Samples taken under non-optimal temperature conditions showed a lower median concentration of 0.77 ng/mL (range: 0-7.4 ng/mL). The four patients who did not respond at days +28 and +180 after RUX initiation (3 with lower gastrointestinal aGvHD, and 1 with ocular, hepatic, and pulmonary cGvHD) showed a median concentration of only (7.4 ng/mL (range, 0-29) ng/mL) with full dosing. The introduction and validation of a liquid chromatography-tandem mass spectrometry method for quantifying plasma RUX concentrations was feasible in our center. Administering predetermined and fixed doses of RUX in patients with SR-GvHD showed highly variable and overlapping plasma drug concentrations. This underscores the potential importance of RUX- pharmacokinetics (PK) monitoring.

Pharmacokinetics and Bioequivalence of Two Formulations of Montelukast Sodium Tablets in Healthy Chinese Volunteers Under Fasting and Fed Conditions.

Montelukast sodium is a leukotriene type 1 receptor antagonist that can be used for the prophylaxis and treatment of asthma. However, the pharmacokinetics of montelukast sodium tablets (10mg) remain unclear in healthy Chinese subjects. Here, a single-dose randomized, open-label, 2-sequence, and 2-period crossover (7-day washout period between treatments) study was performed to compare the pharmacokinetics and bioequivalence between the test products and the reference at a single dose of 10mg among healthy Chinese subjects under fasting and fed conditions. Blood samples were collected at specified time points to analyze the plasma concentrations of montelukast by a validated liquid chromatography-tandem mass spectrometry method. The results showed that the 90% confidence interval values of the geometric mean ratio of test/reference for the maximum plasma drug concentration, area under the concentration-time curve from time 0 to the end, and area under the concentration-time curve from time 0 to infinity were within the range of 80%-125%. Moreover, both the test and reference formulations were safe and well tolerated, with no occurrence of severe adverse events. These results demonstrate that both the test montelukast sodium tablets and the reference showed similar bioequivalence, safety, and tolerability among healthy Chinese subjects under fasting and fed conditions.

Development and validation of a rapid HPLC-MS/MS method for simultaneous determination of cyclosporine A and tacrolimus in whole blood for routine therapeutic drug monitoring in organ transplantation.

Therapeutic drug monitoring is an integral part of organ transplantation. A rapid, simple, economical, and robust high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) method for simultaneously determining the immunosuppressants cyclosporine A and tacrolimus might increase detection efficiency. In this study, we developed and validated a rapid HPLC-MS/MS method. Whole blood samples of 100 μL were prepared by protein precipitation with acetonitrile and 0.5mol. L-1 ZnSO4. Chromatography was performed on a pre-column using a gradient elution with 20mmol. L-1 ammonium formate and 0.1% (v/v) formic acid in water (mobile phase A) and 0.1% (v/v) formic acid in methanol (mobile phase B) at a flow rate of 1.5mL.min-1. The analysis time was 2.2min. Electrospray ionization and multiple reaction monitoring were performed. The lower limit of quantification was set at 1ng. L-1 for tacrolimus and 50 ng. L-1 for cyclosporine A. The method showed adequate accuracy and precision with a sufficient linear range. The calibration curve range of tacrolimus and cyclosporine A was 1-30 and 50-1500 ng·mL-1, respectively. All correlation coefficients were >0.99. The developed HPLC-MS/MS is rapid and can be used for simultaneous monitoring of tacrolimus and cyclosporine A.

Evaluation of protein impurities in Ademetionine 1,4-Butanedisulfonate

Ademetionine 1,4-Butanedisulfonate (SAMe) is widely used as a prescription drug to treat cholestasis associated with liver disease, and is also used to treat depression, Alzheimer's disease and other diseases. Currently, the main way to produce SAMe is by fermentation of S-adenosylmethionine synthetase（SAMS）. However, during the fermentation process, host cells produce contaminants unrelated to the treatment. Among them, host cell protein (HCP), which is co-purified with the drug, is the main impurity in the production process. HCP-induced antigenic reactions can lead to allergies or other adverse reactions and affect drug quality, efficacy, and safety. Enzyme-linked immunosorbent assay (ELISA) and mass spectrometry (MS) are currently used as the main analytical methods for measuring HCP. The ELISA method may ignore some HCPs with low immunogenicity, while mass spectrometry is primarily employed for the characterization of proteomes. Orthogonal methods can more effectively evaluate impurities in drugs. In this study, ELISA was initially utilized to quantify the content of Saccharomyces cerevisiae host protein in 11 batches of SAMe active pharmaceutical ingredient (API) from two suppliers, then the above APIs were qualitatively analyzed by MS method. Following the identification of surrogate peptide of SAMS, an Ultra Performance Liquid Chromatography-Tandem Mass Spectrometry（UPLC-MS/MS）method was established and validated, the SAMS protein residues in 11 batches of samples were subsequently determined. The results demonstrated that the levels of Saccharomyces cerevisiae host protein and SAMS in the samples were both low, with values below 20 ppm and 1.19 ppm, respectively. The qualitative results also indicate that the types of peptide residues in the samples are more diverse than those of protein residues. Furthermore, residues of proteins and peptides can be detected in all samples, which underscores the importance of evaluating the HCP in API. This study employs a range of complementary detection methods to conduct a comprehensive and sensitive evaluation of total HCP and specific proteins in drugs, thereby guiding more informed assessments of the risks posed by HCP impurities in raw materials during pharmaceutical processes.

Trace-level quantification of NDMA in levosulpuride active pharmaceutical ingredient and tablet formulation Using UFLC-MS/MS

Nitrosamine impurities identified in several pharmaceuticals during recent times has raised concerns leading to product recalls worldwide and necessitating sensitive liquid and gas chromatographic methods for trace level detection of nitrosamine impurities. This study developed and validated a ultra-fast liquid chromatography-tandem mass spectrometry (UFLC-MS/MS) method for the quantification of NDMA in Levosulpuride drug substance and tablet formulation. Current method utilizes a triple quadrupole analyzer, atmospheric pressure chemical ionization (APCI) ionization source and multiple reaction monitoring (MRM) scan mode for the analysis. Chromatographic separation was achieved on a Gemini NX-C18 column (150 × 4.6 mm, 3 µm) maintained at 40 °C. The mobile phase consisted of a binary gradient of solvent A (0.1 % formic acid in water) and solvent B (methanol), with a total run time of 18 minutes. Current method achieved excellent linearity, recovery, precision, and sensitivity. Greenness of the developed method was evaluated using the GAPI, AGREE, and AES metrics. Current method is sensitive and selective for NDMA in levosulpuride drug substance and tablet formulations and can be employed for routine quality control analysis in pharmaceutical industry.

A liquid chromatography-tandem mass spectrometry method to determine total matrine residues in edible porcine tissues and its application in a tissue residue depletion study

Matrine (MT), a phytochemical with various anti-infective activities, is expected to become a therapeutic agent against bacterial, viral, and parasitic infections in pigs. However, food safety risks associated with MT have not yet been evaluated. In the present study, a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was developed to determine total MT residues in edible porcine tissues. The tissue samples were hydrolyzed with trypsin to release non-extractable MT. The hydrolysates were extracted with a trichloroacetic acid solution. Tissue extracts were purified using solid-phase extraction and subjected to LC-MS/MS. The method was fully validated according to the United States Food and Drug Administration (USFDA) guidelines and applied to investigate the tissue residue depletion of MT in pigs. Significant accumulation of MT was observed in edible porcine tissues. Notably, MT showed no persistent tissue residue in this study and could be accepted as a safe anti-infective agent in pigs.

Multiparametric LC-MS/MS method for simultaneous determination of eleven antifungal drugs and metabolites in human plasma

A multiparametric liquid chromatography-tandem mass spectrometry method has been developed for the simultaneous quantification of 11 antifungal drugs and their metabolites in human plasma. This method addresses the critical need for therapeutic drug monitoring in the treatment of invasive fungal infections, which are increasingly prevalent among immunocompromised patients and those in intensive care units. The method quantifies flucytosin, fluconazole, itraconazole, hydroxy-itraconazole, posaconazole, isavuconazole, voriconazole, voriconazole-N-oxide, anidulafungin, caspofungin, and micafungin. Key challenges in method development included optimising mass spectrometer settings, chromatographic conditions, and sample preparation techniques to ensure accurate, sensitive, and specific detection. Validation of this method was conducted in accordance with the guidelines set by the USA Food and drug administration and the European Medicines Agency covering linearity, precision, accuracy, selectivity, matrix effect, and stability. The method exhibited robust performance with intra- and inter-assay precision under 10 % and average accuracy for intra- and inter-assay comparison of −2.35 % and 0.80 %, respectively. Limits of detection (0.002 to 0.110 mg/L) and a quantification range between 0.005 and 200 mg/L make this method suitable for clinical TDM applications. The ability to simultaneously analyse eleven antifungals and their metabolites within a single 5-minute run enhances its utility in clinical settings, particularly for critically ill patients who may experience significant pharmacokinetic variations. The method requires only 100 µL of plasma, demonstrating good analytical performances rendering it a valuable tool for optimising antifungal therapy and improving patient outcomes in ICU management.

Mass Spectrometry Probe Combined with Machine Learning to Capture the Relationship between Metabolites and Mitochondrial Complex Activity at the Whole-Cell Level.

Mitochondrial complex activity controls a multitude of physiological processes by regulating the cellular metabolism. Current methods for evaluating mitochondrial complex activity mainly focus on single metabolic reactions within mitochondria. These methods often require fresh samples in large quantities for mitochondria purification or intact mitochondrial membranes for real-time monitoring. Confronting these limitations, we shifted the analytical perspective toward interactive metabolic networks at the whole-cell level to reflect mitochondrial complex activity. To this end, we compiled a panel of mitochondrial respiratory chain-mapped metabolites (MRCMs), whose perturbations theoretically provide an overall reflection on mitochondrial complex activity. By introducing N-dimethyl-p-phenylenediamine and N-methyl-p-phenylenediamine as a pair of mass spectrometry probes, an ultraperformance liquid chromatography-tandem mass spectrometry method with high sensitivity (LLOQ as low as 0.2 fmol) was developed to obtain accurate quantitative data of MRCMs. Machine learning was then combined to capture the relationship between MRCMs and mitochondrial complex activity. Using Complex I as a proof-of-concept, we identified NADH, alanine, and phosphoenolpyruvate as metabolites associated with Complex I activity based on the whole-cell level. The effectiveness of using their concentrations to reflect Complex I activity was further validated in external data sets. Hence, by capturing the relationship between metabolites and mitochondrial complex activity at the whole-cell level, this study explores a novel analytical paradigm for the interrogation of mitochondrial complex activity, offering a favorable complement to existing methods particularly when sample quantities, type, and treatment timeliness pose challenges. More importantly, it shifts the focus from individual metabolic reactions within mitochondria to a more comprehensive view of an interactive metabolic network, which should serve as a promising direction for future research into the functional architecture between mitochondrial complexes and metabolites.

Rapid and Simultaneous Determination of 13 Sulfonamides in Soil by Matrix Solid-Phase Dispersion With High-Performance Liquid Chromatography-Tandem Mass Spectrometry.

Sulfonamides, the most frequent antibacterial agent, are widely used due to their low cost and excellent antibacterial effect. With the emergence of the environment, the potential hazard to the ecological environment has attracted the great attention of humans. Based on matrix solid-phase dispersion coupled with high-performance liquid chromatography-tandem mass spectrometry, an accurate, fast, and sensitive analytical method was developed for the determination of sulfonamides (SAs) in soil. Several influencing factors including the type of dispersants, the ratio of sample-to-sorbent, eluents, and solvent volume were investigated, and the matrix effects were evaluated. Under optimized conditions, the calibration curves exhibited excellent linearity with correlation coefficients (r) exceeding 0.996. The limit of detection (based on signal-to-noise ratio [S/N]=3) and limit of quantification (based on S/N=10) were 0.024-0.058 and 0.079-0.195µg/kg, respectively. The recoveries ranging from 70.12% to 123.63% were obtained with a relative standard deviation of less than 15% at three levels (20, 40, and 200µg/kg). The developed method was successfully applied to analyze 13 SAs at trace levels in a real soil sample. This proposed method would be an alternative and suitable for routine application in the future owing to its rapidity, sensitivity, and affordability.