Ultra-high Performance Liquid Chromatography-tandem Mass Research Articles

Anti-diabetic and anti-inflammatory bioactive hits from Coriaria intermedia Matsum. stem and Dracontomelon dao (Blanco) Merr. & Rolfe bark through bioassay-guided fractionation and liquid chromatography-tandem mass spectrometry.

Women have been found to be at a higher risk of morbidity and mortality from type 2 diabetes mellitus (T2DM) and asthma. α-Glucosidase inhibitors have been used to treat T2DM, and arachidonic acid 15-lipoxygenase (ALOX15) inhibitors have been suggested to be used as treatments for asthma and T2DM. Compounds that inhibit both enzymes may be studied as potential treatments for people with both T2DM and asthma. This study aimed to determine potential anti-diabetic and anti-inflammatory bioactive hits from Coriaria intermedia Matsum. stem and Dracontomelon dao (Blanco) Merr. & Rolfe bark. A bioassay-guided fractionation framework was used to generate bioactive fractions from C. intermedia stem and D. dao bark. Subsequently, dereplication through ultra-high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) and database searching was performed to putatively identify the components of one bioactive fraction from each plant. Seven compounds were putatively identified from the C. intermedia stem active fraction, and six of these compounds were putatively identified from this plant for the first time. Nine compounds were putatively identified from the D. dao bark active fraction, and seven of these compounds were putatively identified from this plant for the first time. One putative compound from the C. intermedia stem active fraction (corilagin) has been previously reported to have inhibitory activity against both α-glucosidase and 15-lipoxygenase-1. It is suggested that further studies on the potential of corilagin as an anti-diabetic and anti-inflammatory treatment should be pursued based on its several beneficial pharmacological activities and its low reported toxicity.

Determination of higenamine in common natural spices by ultrahigh-performance liquid chromatography-tandem mass spectrometry.

Higenamine (HG) is a β2 receptor agonist and was explicitly added to the Prohibited List of the World Anti-Doping Agency in 2017. This compound is prohibited in both in- and out-of-competition athletes and falls under the category of nonthreshold substances. Because of HG presence in numerous plants, as evidenced by a growing body of research data, an exception was made for HG in the TD2017MRPL document, in which adverse analytical findings (AAFs) were not reported if the urinary HG concentration was less than 10ng/mL. In this study, a comprehensive and systematic analysis of the HG content in five batches of samples from each of the 48 natural spices selected for this investigation was conducted using UPLC-MS/MS technology. Method validation was carried out in accordance with the ICH Analytical Procedures and Methods Validation for Drugs and Biologics Guidance, and the experimental results demonstrated that the method provided appropriate sensitivity, precision, stability, linearity, and accuracy. HG was detected for the first time in Houttuynia cordata, Zingiber officinale, Cinnamomum cassia, Stevia rebaudiana, Piper nigrum, Siraitia grosuenorii, Platycodon grandiflorus, and Myristica fragrans. Furthermore, the content of HG was found to vary significantly among the different plant parts of Nelumbo nucifera, such as rhizomes, leaves, seeds, and plumules. This paper provides systematic and comprehensive data to support the safe use of spices in athletes' diets, thereby reducing the risk of food-sourced doping violations.

Hawthorn leaf flavonoids alleviate the deterioration of atherosclerosis by inhibiting SCAP-SREBP2-LDLR pathway through sPLA2-ⅡA signaling in macrophages in mice

Ethnopharmacological relevanceHawthorn leaves are a combination of the dried leaves of the Rosaceae plants, i.e., Crataegus pinnatifida Bge. or Crataegus pinnatifida Bge. var. major N. E. Br., is primarily cultivated in East Asia, North America, and Europe. hawthorn leaf flavonoids (HLF) are the main part of extraction. The HLF have demonstrated potential in preventing hypertension, inflammation, hyperlipidemia, and atherosclerosis. However, the potential pharmacological mechanism behind its anti-atherosclerotic effect has yet to be explored. Aim of the studyThe in vivo and in vitro effects of HLF on lipid-mediated foam cell formation were investigated, with a specific focus on the levels of secreted phospholipase A2 type IIA (sPLA2-II A) in macrophage cells. Materials and methodsThe primary constituents of HLF were analyzed using ultra-high performance liquid chromatography and liquid chromatography-tandem mass spectrometry. In vivo, HLF, at concentrations of 5 mg/kg, 20 mg/kg, and 40 mg/kg, were administered to apolipoprotein E knockout mice (ApoE−/−) fed by high-fat diet (HFD) for 16 weeks. Aorta and serum samples were collected to identify lesion areas and lipids through mass spectrometry analysis to dissect the pathological process. RAW264.7 cells were incubated with oxidized low-density lipoprotein (ox-LDL) alone, or ox-LDL combined with different doses of HLF (100, 50, and 25 μg/ml), or ox-LDL plus 24-h sPLA2-IIA inhibitors, for cell biology analysis. Lipids and inflammatory cytokines were detected using biochemical analyzers and ELISA, while plaque size and collagen content of plaque were assessed by HE and the Masson staining of the aorta. The lipid deposition in macrophages was observed by Oil Red O staining. The expression of sPLA2-IIA and SCAP-SREBP2-LDLR was determined by RT-qPCR and Western blot analysis. ResultsThe chemical profile of HLF was studied using UPLC-Q-TOF-MS/MS, allowing the tentative identification of 20 compounds, comprising 1 phenolic acid, 9 flavonols and 10 flavones, including isovitexin, vitexin-4″-O-glucoside, quercetin-3-O-robibioside, rutin, vitexin-2″-O-rhamnoside, quercetin, etc. HLF decreased total cholesterol (TC), triglycerides (TG), low-density lipoprotein cholesterol (LDL-C), and non-high-density lipoprotein cholesterol (non-HDL-C) levels in ApoE−/− mice (P < 0.05), reduced ox-LDL uptake, inhibited level of inflammatory factors, such as IL-6, IL-8, TNF-α, and IL-1ꞵ (P < 0.001), and alleviated aortic plaques with a thicker fibrous cap. HLF effectively attenuated foam cell formation in ox-LDL-treated RAW264.7 macrophages, and reduced levels of intracellular TC, free cholesterol (FC), cholesteryl ester (CE), IL-6, TNF-α, and IL-1β (P < 0.001). In both in vivo and in vitro experiments, HLF significantly downregulated the expression of sPLA2-IIA, SCAP, SREBP2, LDLR, HMGCR, and LOX-1 (P < 0.05). Furthermore, sPLA2-IIA inhibitor effectively mitigated inflammatory release in RAW264.7 macrophages and regulated SCAP-SREBP2-LDLR signaling pathway by inhibiting sPLA2-IIA secretion (P < 0.05). ConclusionHLF exerted a protective effect against atherosclerosis through inhibiting sPLA2-IIA to diminish SCAP-SREBP2-LDLR signaling pathway, to reduce LDL uptake caused foam cell formation, and to slow down the progression of atherosclerosis in mice.

Discovering novel biomarkers for diagnosis and treatment monitoring of active pulmonary tuberculosis by ion metabolism analysis

Tuberculosis (TB) is a highly lethal infectious disease that poses a global threat. Timely and accurate biomarker for TB diagnosis and treatment monitoring remains a pressing need. Ions, the crucial trace element for humans, may be potential targets for TB diagnosis and the forecasting of TB development. To explore the potential of ions as biomarkers, we measured and compared the levels of various ions in whole blood and plasma samples from healthy control (HC), pulmonary TB patients (TB), cured pulmonary TB patients (RxTB), and other non-TB pneumonia patients (PN) by using ultra-high performance liquid chromatography-tandem mass spectrometry. Our study demonstrated that Cu (AUC = 0.670), Pb (AUC = 0.660), and Zn (AUC = 0.701) in whole blood exhibited promising diagnostic performance for TB. Then we used a neural network (NNET) for TB prediction, the AUC values used to differentiate definite TB from HC or PN in plasma were 0.867 and 0.864, respectively. The AUC values used to differentiate definite TB from HC or PN in whole blood were 0.818 and 0.660, respectively. Our correlation analysis showed that Zn (r= 0.356, p=0.001) and Cu (r= 0.361, p=0.0004) in plasma are most closely related to disease severity. Additionally, six ions (Cu, Sb, V, Mn, Fe, Sr) in plasma and whole blood were altered following anti-TB therapy. These results showed that ions could be diagnostic biomarkers for TB. Furthermore, the level of particular ions can forecast the degree of lung damage and the success of the TB treatment. In conclusion, this study highlights the possibility of using ions from blood samples to enable rapid tuberculosis diagnosis.

Determination of nitrofuran metabolites and nifurpirinol in animal tissues and eggs by ultra-high performance liquid chromatography-tandem mass spectrometry validated according to Regulation (EU) 2021/808

In this work, an analytical method for the determination of eight non-allowed nitrofurans, including nifurpirinol and the metabolites of furazolidone, furaltadone, nitrofurantoin, nitrofurazone, nifursol, nitrovin, and nifuroxazide in animal tissues, including muscle (poultry, bovine, ovine, porcine, rabbit, and fish), kidney (bovine, ovine, porcine), and eggs, has been developed and validated according to Regulation (EU) 2021/808. The method was based on derivatization with 2-nitrobenzaldehyde in acid medium, followed by vortex-assisted liquid-liquid extraction and solid phase extraction for sample purification prior to ultra-high performance liquid chromatography-tandem mass spectrometry. Under selected conditions, the method was validated showing satisfactory relative matrix effects (CV ≤ 20 %), linearity (R2 ≥ 0.98), trueness (≤20 %, expressed as bias), accuracy (83–120 %), repeatability (1.7–19.9 %), reproducibility (1.9–25.7 %), specificity (blank signal ≤30 % at the LCL), and ruggedness. The decision limit for confirmation (CCα) for the target analytes ranged from 0.27 to 0.35 μg kg−1, all below the current reference point for action (RPA) of 0.5 μg kg−1 for the studied compounds. This validated method is currently accredited according to UNE-EN ISO/IEC 17025 by the Spanish National Accreditation Body (ENAC) to be implemented for official control analyses in the Public Health Laboratory of Valencia (Spain).

Development of an analytical system for dried blood spots for forensic toxicology: a case study of five common drugs and poisons

Dried blood spot (DBS) technology is a simple and convenient method for collecting, transporting, and storing blood samples on filter paper, and has numerous applications in the clinical, research, and public health settings. This technique is gaining popularity in the field of forensic science because it facilitates the rapid analysis of prohibited drugs in blood samples and offers significant advantages in toxicology scenarios such as drinking-driving screening, drug abuse detection, and doping detection. However, the lack of a standardized system and the fact that its stability and reliability have not been thoroughly researched and demonstrated limit its application in judicial practice in China. DBS samples can be prepared, stored, and analyzed in various ways, all of which may significantly affect the results. In this study, we developed a method based on ultra-high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) that focuses on the preparation, pretreatment, analysis, and storage of DBS samples. A thorough investigation was conducted to examine the optimal preparation conditions, including the blood spot matrix, drying technique, and preprocessing parameters, such as the solvent and extraction method. Moreover, the analytical conditions, such as the mobile phase system and elution gradient, were established to facilitate the quantitative detection of methamphetamine, lidocaine, ketamine, fentanyl, and diazepam in both DBS and whole-blood samples. The impact of storage conditions, such as the temperature, humidity, and sealing, on the analytical results of the DBS and whole-blood samples was also examined. The results showed a strong linear relationship for lidocaine and fentanyl within the range of 0.5-100 ng/mL. Similarly, methamphetamine, ketamine, and diazepam exhibited good linearity within the range of 2-100 ng/mL. The coefficients of determination (r2) ranged from 0.9983 to 0.9997, and the limits of detection ranged from 0.2 to 0.5 ng/mL, indicating a high degree of correlation and sensitivity. Stability tests demonstrated that the five target substances remained stable in the DBS for 60 days, with the measured contents deviating from the nominal values by 15%. Moreover, the measurement results of the DBS samples were highly similar to those of the whole-blood samples, with mean percentage differences of 4.44%, 3.50%, 7.66%, 5.10%, and 5.25% for fentanyl, diazepam, ketamine, lidocaine, and methamphetamine, respectively. Throughout the 60-day storage period, the maintenance of temperatures of -20 and 4 ℃, as well as sealing and dry storage, was not necessary. Room temperature was the most practical storage environment for the DBS samples. The results for each target showed very small concentration differences between the whole-blood and DBS samples, indicating that the DBS samples were suitable for drug and poison analysis in blood. Furthermore, the DBSs exhibited high quantitative consistency with the whole-blood samples, rendering them suitable matrices for preserving blood samples. Because DBS samples are easy to handle and store, they can realize the lightweight preservation of blood samples and provide a novel solution for the analysis and preservation of blood samples in public security practice. We recommend conducting comprehensive validations before utilizing DBS for analysis, particularly in terms of quantification, to ensure the judicial reliability of the results.

Comparative pharmacokinetics of six components in normal and rheumatoid arthritis rats after intragastrical administration of Qianghuo Shengshi Decoction granules by LC-MS/MS

ObjectiveTo investigate the plasma pharmacokinetics of six representative components (nodakenin, osthole, 5-O-methylvisammioside, ferulic acid, liquiritigenin, and liquiritin), which were the ingredients of Qianghuo Shengshi Decoction (QSD) granules, in normal and rheumatoid arthritis (RA) rats administrated QSD granules intragastrically. MethodsA rapid and accurate ultra-high performance liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was developed for the simultaneous determination of six components in plasma, and it showed a good specificity, linearity, intra-day and inter-day precision, intra-day and inter-day accuracy, extraction recovery, stability, and the less matrix effect. ResultsThe validated LC-MS/MS method was successfully used to compare the plasma pharmacokinetics of six ingredients between normal and RA rats after intragastrical administration of QSD granules and differences in the pharmacokinetics were found in two types of rats. The absorption rate in the RA rats was lower for nodakenin, osthole, 5-O-methylvisammioside, liquiritigenin and liquiritin than in the normal group, while the absorption rate of ferulic acid remained constant in two groups. Incomparisonwith the normal rats, the exposure concentration of nodakenin was higher and that of other five components except for nodakenin was lower under pathological conditions. Additionally, the absorptive amount of nodakenin, osthole, 5-O-methylvisammioside and liquiritin was increased and that of ferulic acid and liquiritigenin was reduced in the RA rats than in the normal rats. Compared with the normal rats, the retention time of nodakenin, ferulic acid and liquiritin was reduced in vivo, whereas the retention time of osthole, 5-O-methylvisammioside and liquiritigenin was raised in the body for the RA rats. In contrast to the normal rats, the data demonstrated an increase in the elimination velocity of nodakenin and a decrease in the elimination velocity of the other five components except for nodakenin in the pathological state. ConclusionThis study showed that the pharmacokinetic behavior of the six components, nodakenin, osthole, 5-O-methylvisammioside, ferulic acid, liquiritigenin, and liquiritin, is different in vivo between normal and pathological states of rats, and this research provided the necessary experimental data to explain the pharmacokinetics of QSD granules in both normal and pathological states and provide some references for its clinical application at some level.

Development and validation of an ultra-high performance liquid chromatography-tandem mass spectrometry method to quantify antiretroviral drug concentrations in human plasma for therapeutic monitoring

Antiretroviral therapy (ART) is highly effective for the treatment of HIV-1 infection. ART previously consisted of concomitant administration of many drugs, multiple times per day. Currently, ART generally consists of two- or three-drug regimens once daily as fixed-dose combinations. Drug monitoring may be necessary to ensure adequate concentrations are achieved in the plasma over the dosing interval and prevent further HIV resistance formation. Additionally, nonadherence remains an issue, highlighting the need to ensure sufficient ART exposure. Towards this effort, we developed and validated a highly selective ultra performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) method for the simultaneous quantification of a panel of nine antiretrovirals: abacavir, bictegravir, cabotegravir, dolutegravir, doravirine, emtricitabine, lamivudine, raltegravir, and tenofovir in human plasma. Using only 50µL of plasma, a simple protein precipitation with acetonitrile with internal standards followed by reconstitution in 50uL (high) or 400 uL (low)was performed. Analyte separation was achieved using a multistep UPLC gradient mixture of (A: 0.1% formic acid in water and B: acetonitrile) and a Waters CORTECS T3 (2.1×100mm) column. The method was comprehensively validated according to the United States Food and Drug Administration Bioanalytical Guidelines over two clinically relevant ranges (1-250ng/mL and 100-5000ng/mL) with excellent linearity (R2 > 0.99 for all). The assay run time was 7.5minutes. This method achieves acceptable performance of trueness (89.7-104.1%), repeatability, and precision (CV <15%), and allows for simultaneous quantification of guideline-recommended ART regimens. This method can be utilized for the therapeutic monitoring of antiretrovirals in human plasma.

Simultaneous determination of 28 illegal drugs in sewage by high throughput online SPE-ISTD-UHPLC-MS/MS

This study developed an online solid-phase extraction ultra-high performance liquid chromatography-tandem mass spectrometry (Online-SPE-UHPLC-MS/MS) method for the analysis of 28 illegal drugs in sewage. To achieve this, 28 isotope internal standards (ISTDs) were added to 3 mL sewage samples, the pH was adjusted to 7–8 using hydrochloric acid or 20% ammonia water, followed by centrifugation, filtration, and analysis using UHPLC-MS/MS. The results indicated an excellent linearity of 1–300 ng L−1, and cotinine in the concentration range of 20–6000 ng L−1, linear correlation coefficient R2 > 0.995, with the limit of detection (LOD) of 0.01–6 ng L−1, and a limit of quantification (LOQ) of 0.1–20 ng L−1. The addition of three concentrates of low (2 ng L−1/40 ng L−1), medium (20 ng L−1/400 ng L−1), and high concentration (200 ng L−1/4000 ng L−1) demonstrated the matrix effect of the target compound between ± 22.0%. The extraction recovery was 70.0–119.4%, and a percent accuracy of 75.7–118.1%. Similarly, the intra- and inter-day precisions were 1.8–20.0% and 1.5–18.9%, respectively. The results cemented the sensitivity, accuracy, reliability, strong specificity, and reproducibility, which can be used to screen 28 illegal drugs in sewage for trace analysis.

Comparison of a Daily Steviol Glycoside Beverage compared with a Sucrose Beverage for Four Weeks on Gut Microbiome in Healthy Adults

BackgroundRecent studies suggest that some nonnutritive sweeteners (NNS) have deleterious effects on the human gut microbiome (HGM). The effect of steviol glycosides on the HGM has not been well studied. ObjectiveWe aimed to evaluate the effects of stevia- compared with sucrose-sweetened beverages on the HGM and fecal short-chain fatty acid (SCFA) profiles. MethodsUsing a randomized, double-blinded, parallel-design study, n = 59 healthy adults [female/male, n = 36/23, aged 31±9 y, body mass index (BMI): 22.6±1.7 kg/m2] consumed 16 oz of a beverage containing either 25% of the acceptable daily intake (ADI) of stevia or 30 g of sucrose daily for 4 weeks followed by a 4-week washout. At weeks 0 (baseline), 4, and 8, the HGM was characterized via shotgun sequencing, fecal SCFA concentrations were measured using ultra-high performance liquid chromatography-tandem mass spectrometry and anthropometric measurements, fasting serum glucose, insulin and lipids, blood pressure, pulse, and 3-d diet records were obtained. ResultsThere were no significant differences in the HGM or fecal SCFA between the stevia and sucrose groups at baseline (P > 0.05). At week 4 (after intervention), there were no significant differences in the HGM at the phylum, family, genus, or species level between the stevia and sucrose groups and no significant differences in fecal SCFA. At week 4, BMI had increased by 0.3 kg/m2 (P = 0.013) in sucrose compared with stevia, but all other anthropometric and cardiometabolic measures and food intake did not differ significantly (P > 0.05). At week 8 (after washout), there were no significant differences in the HGM, fecal SFCA, or any anthropometric or cardiometabolic measure between the stevia and sucrose groups (P > 0.05). ConclusionsDaily consumption of a beverage sweetened with 25% of the ADI of stevia for 4 weeks had no significant effects on the HGM, fecal SCFA, or fasting cardiometabolic measures, compared with daily consumption of a beverage sweetened with 30 g of sucrose. Trial registrationclinicaltrials.gov as NCT05264636

Quantitative Analysis of Cenobamate and Concomitant Anti-Seizure Medications in Human Plasma via Ultra-High Performance Liquid Chromatography-Tandem Mass Spectrometry.

Cenobamate (CNB) is a new anti-seizure medication (ASM) recently introduced in clinical practice after approval by the FDA and EMA for the add-on treatment of focal onset seizures in adult patients. Although its mechanism of action has not been fully understood, CNB showed promising clinical efficacy in patients treated with concomitant ASMs. The accessibility of CNB could pave a way for the treatment of refractory or drug-resistant epilepsies, which still affect at least one-third of the patients under pharmacological treatment. In this context, therapeutic drug monitoring (TDM) offers a massive opportunity for better management of epileptic patients, especially those undergoing combined therapy. Here, we describe the first fully validated ultra-high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) method for the quantification of CNB and concomitant ASMs in human plasma, with samples extracted either manually or by means of a liquid handler. Our method was validated according to the most recent ICH International Guideline M10 for Bioanalytical Method Validation and Study Sample Analysis. The method proved to be selective for CNB and displayed a linear range from 0.8 to 80 mg/L; no matrix effect was found (98.2 ± 4.1%), while intra-day and inter-day accuracy and precision were within the acceptance range. Also, CNB short- and long-term stability in plasma under different conditions was assessed. Leftover human plasma samples were employed as study samples for method validation. Our method proved to be highly sensitive and selective to quantify CNB and concomitant ASMs in human plasma; therefore, this method can be employed for a routinely TDM-based approach to support physicians in the management of an epileptic patient.

Detection of antibiotic residues in groundwater with a validated multiresidue UHPLC-MS/MS quantification method

The occurrence of antibiotic residues in the environment has received considerable attention because of their potential to select for bacterial resistance. The overuse of antibiotics in human medicine and animal production results in antibiotic residues entering the aquatic environment, but concentrations are currently not well determined. This study investigates the occurrence of antibiotics in groundwater in areas strongly related to agriculture and the antibiotic treatment of animals. A multiresidue method was validated according to EU Regulation 2021/808, to allow (semi-)quantitative analysis of 78 antibiotics from 10 different classes: β-lactams, sulfonamides, tetracyclines, lincosamides, amphenicols, (fluoro)quinolones, macrolides, pleuromutilins, ansamycins and diaminopyrimidines using ultra-high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS). This method was used to test different storage conditions of these water samples during a stability study over a period of 2 weeks. Sulfonamides, lincosamides and pleuromutilins were the most stable. Degradation was most pronounced for β-lactam antibiotics, macrolides and ansamycins. To maintain stability, storage of samples at −18 °C is preferred. With the validated method, antibiotic residues were detected in groundwater, sampled from regions associated with intensive livestock farming in Flanders (Belgium). Out of 50 samples, 14% contained at least one residue. Concentrations were low, ranging from < LOD to 0.03 μg/L. Chloramphenicol, oxolinic acid, tetracycline and sulfonamides (sulfadiazine, sulfadoxine, sulfamethazine and sulfisoxazole) were detected. This study presents a new method for the quantification of antibiotic residues, which was applied to investigate the presence of antibiotic residues in groundwater in Flanders.

Simultaneous determination of 58 glucocorticoid residues in milk by ultra-high performance liquid chromatography-tandem mass spectrometry

Abuse of glucocorticoid veterinary drugs in dairy industry can potentially threat milk safety and consequently influence human health. Here a reliable method for determination of 58 glucocorticoid drug residues in milk was established by combining solid phase extraction with ultra-high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS). The analytes were extracted with acetonitrile and cleanup with EMR-Lipid lipid removal column. The analytes were chromatographically separated using Poroshell EC-C18 column and acquired by electrospray ionization with multiple-reaction monitoring (MRM) mode. The limit of quantification (S/N ≥ 10) ranged from 0.2 to 2.0 µg/kg and the limit of detection (S/N ≥ 3) ranged from 0.1 to 1.0 µg/kg. Average recoveries were from 71% to 113%, the relative standard deviations (RSDs) were less than 15%, and the correlation coefficients (R2) of calibration curves exceeded 0.99. The method was applied to detect twenty milk products obtained from local supermarkets including ten pasteurized milk and ten UHT milk. Two endogenous glucocorticoids, i.e. hydrocortisone and cortisone were detected but not exceed the maximum residue limits (MRLs).

A New Ultra High-Performance Liquid Chromatography-Tandem Mass Spectrometry Method to Monitor 1-Amino-4-Methyl-Piperazine and 1-Methyl-4-Nitroso-Piperazine in Rifampicin Hydrolysis: A Standard Addition Approach to Quantify Nitrosamine in Rifampicin

1-amino-4-methyl-piperazine (AMP) is both a starting material for the synthesis of rifampicin (RIF) and a degradation product of RIF hydrolysis. 1-methyl-4-nitroso-piperazine (MNP) is an oxidation product of AMP as well as a potentially genotoxic N-nitrosamine. The EMA and FDA have approved an ad interim limit of 5 ppm for MNP in RIF drug products. As in-house methods for the analysis of MNP in RIF use a wide range of conditions for mobile phases and sample diluents, we decided to investigate whether these conditions affect the formation of MNP and AMP. A UHPLC-MS/MS method was developed to simultaneously quantify AMP and MNP during RIF hydrolysis in buffered aqueous solutions at different pH levels. Analyses were performed in MRM mode; separations were carried out on an InfinityLab Poroshell HPH-C18 (100 mm, 2.1 mm i.d., particle size 1.9 μm). In aqueous RIF solutions, the content of AMP and MNP increases with time; at different pHs, the concentration of AMP increases much faster in acidic than in basic solutions; and the increase in MNP can be reduced by the addition of ascorbic acid. To avoid an overestimation of MNP, water should not be used as a diluent in RIF sample preparations. Methanol is a more suitable diluent than water. A standard addition method has been validated for the quantification of MNP in RIF drug substances.

Isolation and identification of active components from Grifola frondosa and its anti-EV71 virus effect.

It is reported that anti-enterovirus 71 (EV71) drugs have some side effects on human health. Notably, fungi plays a crucial role in promoting human health and anti-virus. Grifola frondosa is a type of large medicinal and edible fungi, rich in active substances. The present study aimed to investigate the anti-EV71 effect of G. frondosa and the potential active substances. In the present study, the water extract of G. frondosa was subjected to ethanol precipitation to obtain the water-extracted supernatant of G. frondosa (GFWS) and water-extracted precipitation of G. frondosa. Their inhibitory effects on EV71 virus were studied based on a cell model. The results showed that GFWS had stronger security and anti-EV71 effects. In addition, the chemical constituents of GFWS were identified by ultra-high performance liquid chromatography-tandem mass spectrometry, which were selected for further separation and purification. Three compounds, N-butylaniline, succinic acid and l-tryptophan, were isolated from GFWS by NMR spectroscopy. It is noteworthy that N-butylaniline and l-tryptophan were isolated and identified from the G. frondosa fruiting bodies for the first time. Our study found that l-tryptophan has anti-EV71 virus activity, which reduced EV71-induced apoptosis and significantly inhibited the replication process after virus adsorption. Furthermore, it could also bind to capsid protein VP1 to prevent the virus from attaching to the cells. l-tryptophan was an inhibitor of the EV71 virus, which could be used in infant nutrition and possibly provide a new drug to treat hand, foot and mouth disease. © 2024 Society of Chemical Industry.

Evaluation of drug interactions of Saposhnikoviae Radix and its major components with astragaloside IV and paeoniflorin using in vitro and in vivo experiments

Saposhnikoviae Radix (SR) may enhance the pharmacodynamics of Huangqi Chifeng Tang (HQCFT) in the treatment of cerebral infarction according to our previous research, but the underlying mechanism is unknown. Herein, an in vivo pharmacokinetic assay in rats and in vitro MDCK-MDR1 cell assays were used to investigate the possible mechanism of SR, its main components, and its interactions with Astragali Radix (AR) and Paeoniae Radix (PR). An ultrahigh-performance liquid chromatography–tandem mass spectrometry (UPLC‒MS/MS)-based analytical method for quantifying astragaloside IV (ASIV) and paeoniflorin (PAE) in microdialysis and transport samples was developed. The pharmacokinetic parameters of SR were determined using noncompartmental analyses CCK-8 assays were used to detect the cytotoxicity of ASIV, PAE, cimifugin (CIM), prim-o-glucosylcimifugin (POG) and their combinations. Moreover, drug transport was studied using MDCK-MDR1 cells. Western blotting was performed to measure the protein expression levels of P-GP and MRP1. Claudin-5, ZO-1, and F-actin expression was determined via immunohistochemical staining of MDCK-MDR1 cells. harmacokinetic studies revealed that, compared with those of Huangqi Chifeng Tang-Saposhnikoviae Radix (HQCFT-SR), the Tmax of ASIV increased by 11.11 %, and the MRT0-t and Tmax of PAE increased by 11.19 % and 20 %, respectively, in the HQCFT group. Transport studies revealed that when ASIV was coincubated with 28 μM CIM or POG, the apparent permeability coefficient (Papp) increased by 71.52 % and 50.33 %, respectively. Coincubation of PAE with 120 μM CIM or POG increased the Papp by 87.62 % and 60.95 %, respectively. Moreover, CIM and POG significantly downregulated P-gp and MRP1 (P < 0.05), inhibited the expression of Claudin-5, ZO-1, and F-actin (P < 0.05), and affected intercellular tight junctions (TJs). In conclusion, our study successfully established a selective, sensitive and reproducible UPLC‒MS/MS analytical method to detect drug‒drug interactions between SR, AR and PR in vivo and in vitro, which is beneficial for enhancing the therapeutic efficacies of AR and PR. Moreover, this study provides a theoretical basis for further research on the use of SR as a drug carrier.

Risk analysis of serum chemical residues for metabolic associated fatty liver disease based on exposome-lipidome wide association study

Metabolic associated fatty liver disease (MAFLD) is a common liver disease with a prevalence of up to 25%; it not only adversely affects human health but also aggravates the economic burden of society. An increasing number of studies have suggested that the occurrence of chronic noncommunicable diseases is affected by both environmental exposures and genetic factors. Research has also shown that environmental pollution may increase the risk of MAFLD and promote its occurrence and development. However, the relationship between these concepts, as well as the underlying exposure effects and mechanism, remains incompletely understood. Lipidomics, a branch of metabolomics that studies lipid disorders, can help researchers investigate abnormal lipid metabolites in various disease states. Lipidome-exposome wide association studies are a promising paradigm for investigating the health effects of cumulative environmental exposures on biological responses, and could provide new ideas for determining the associations between metabolic and lipid changes and disease risk caused by chemical-pollutant exposure. Hence, in this study, targeted exposomics and nontargeted lipidomics studies based on ultra-high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) and ultra-high performance liquid chromatography-high resolution mass spectrometry (UHPLC-HRMS) were used to characterize exogenous chemical pollutants and endogenous lipid metabolites in the sera of patients with MAFLD and healthy subjects. The results demonstrated that fipronil sulfone, malathion dicarboxylic acid, and monocyclohexyl phthalate may be positively associated with the disease risk of patients diagnosed as simple fatty liver disease (hereafter referred to as MAFLD(0)). Moreover, fipronil sulfone, acesulfame potassium, perfluorooctanoic acid (PFOA), perfluorononanoic acid (PFNA), perfluoroundecanoic acid (PFUnDA), 4-hydroxybenzophenone, and 3,5-di-tert-butyl-4-hydroxybenzoic acid (DBPOB) may be positively associated with the disease risk of patients diagnosed as fatty liver complicated by single or multiple metabolic disorders. Association analysis was carried out to explore the lipid metabolites induced by chemical residues. Triglyceride (TG) and diglyceride (DG) were significantly increased in MAFLD and MAFLD(0). The numbers of carbons of significantly changed DGs and TGs were mainly in the ranges of 32-40 and 35-60, respectively, and both were mainly characterized by changes in polyunsaturated lipids. Most of the lipid-effect markers were positively correlated with chemical residues and associated with increased disease risk. Our research provides a scientific basis for studies on the association and mechanism between serum chemical-pollutant residues and disease outcomes.

Determination of 55 Veterinary Antibiotics in Chicken Manure Using Ultra-High-Performance Liquid Chromatography – Tandem Mass Spectrometry (UPLC-MS/MS)

A highly efficient method for the detection and quantification of 55 veterinary antibiotics (VAs) in chicken manure belonging to six drug classes using solid-phase extraction (SPE) and ultrahigh-performance liquid chromatography–tandem mass spectrometry (UPLC–MS/MS) was developed. The mixture of EDTA–McIlvaine buffer (pH 3.0) and the organic extractant (methanol–acetonitrile, 1:3, v/v) was used to isolate the target VAs from freeze-dried chicken manure. The extract was purified by a hydrophile–lipophile balance cartridge. All antibiotics showed excellent linear relationships in the range between 1 and 100 µg/kg with the coefficients of determination of the standard curves above 0.990. The recoveries of antibiotics were between 35.66% and 103.54% using matrix-matched calibration for quantification. The limits of detection and quantification were from 0.01 to 1.59 µg/kg and 0.04 to 4.76 µg/kg, respectively. The method was demployed to determine VAs in chicken manure from 12 farms, revealing contamination in all samples. A total of 13 VAs belonging to six classes were detected with concentrations up to 41.47 mg/kg. The presence of high concentrations of antibiotic residues in poultry manure poses a potential risk for environmental contamination. This work has identified significant differences in the types of VAs compared to literature reports. Additionally, it detected pleuromutilin antibiotics in chicken manure for the first time.

Comparison of filter membranes in the analysis of 183 veterinary and other drugs by liquid chromatography-tandem mass spectrometry.

Although filtration is one of the most common steps in sample preparation for chemical analysis, filter membrane materials can leach contaminants and/or retain some analytes in the filtered solutions. In multiclass, multiresidue analysis of veterinary drugs, it is challenging to find one type of filter membrane that does not retain at least some of the analytes before injection in ultrahigh-performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS). In this study, different filter membranes were tested for use in UHPLC-MS/MS analysis of 183 diverse drugs in bovine muscle, kidney, and liver tissues. Membranes evaluated consisted of polytetrafluoroethylene (PTFE), polyvinylidene difluoride (PVDF), polyethersulfone, nylon, and regenerated cellulose. Drug classes represented among the analytes included β-agonists, β-lactams, anthelmintics, macrolides, tetracyclines, sulfonamides, tranquilizers, (fluoro)quinolones, anti-inflammatories, nitroimidazoles, coccidiostats, phenicols, and others. Although the presence of a matrix helped reduce the binding of analytes on surface active sites, all of the filter types partially retained at least some of the drugs in the final extracts. In testing by flow-injection analysis, all of the membrane filters were also observed to leach interfering components. Ultimately, filtration was avoided altogether in the final sample preparation approach known as the quick, easy, cheap, effective, rugged, safe, efficient, and robust (QuEChERSER) mega-method, and ultracentrifugation was chosen as an alternative.

Analytical Validation of a Volumetric Absorptive Microsampling Method for Therapeutic Drug Monitoring of the Oral Targeted Anticancer Agents, Abiraterone, Alectinib, Cabozantinib, Imatinib, Olaparib, and Sunitinib, and Metabolites.

Volumetric Absorptive Microsampling (VAMS) is a useful tool for therapeutic drug monitoring (TDM) of oral targeted anticancer agents. VAMS aims to improve safety and efficacy by enabling at-home blood sample collection by patients. This study aimed to develop and validate an ultra-high performance liquid chromatography-tandem mass spectrometry method for the quantitative determination of abiraterone, alectinib, cabozantinib, imatinib, olaparib, sunitinib, and the metabolites, Δ(4)-abiraterone (D4A), alectinib-M4, imatinib-M1, and N-desethyl sunitinib, in dried whole blood samples using VAMS to support TDM. After the collection of 10 μL of whole blood sample using the VAMS device, the analytes were extracted from the tip using methanol with shaking, evaporated, and reconstituted in acetonitrile:0.1 mol/L ammonium hydroxide in water (1:1, vol/vol). The extracts were then analyzed using ultra-high performance liquid chromatography-tandem mass spectrometry. Validation experiments based on the ICH M10 guideline were carried out, and stability was evaluated under shipping and storage conditions. VAMS specimens were collected in the outpatient clinic to demonstrate the applicability of the assay. The validated range of the method was considered accurate and precise for all analytes. Accordingly, the validation experiments met the relevant requirements, except for cross-analyte interference. Based on the stability data, shipment can be performed at room temperature within 14 days after sample collection and the VAMS specimen can be stored up to 9 months at -20 and -70°C. Samples from 59 patients were collected at the hospital. The developed method could be used to successfully quantify the concentrations of abiraterone, D4A, alectinib, alectinib-M4, cabozantinib, imatinib, imatinib-M1, olaparib, sunitinib, and N-desethyl sunitinib within the validated range using VAMS. Therefore, the method can be used to estimate the dried whole blood-to-plasma ratios for TDM in the clinic.