Performance Liquid Chromatography-tandem Mass Spectrometer Research Articles

Validation and Application of UPLC-MS/MS Method to Analysis of Glyphosate and its Metabolites in Water.

A method was developed to determine glyphosate and their metabolites in water. The widespread use of this herbicide in agricultural activities worldwide, despite the reported adverse effects on both the environment and health, is a cause for concern and makes it necessary to monitor its presence through a method that guarantees the determination at trace levels. A direct extraction of the analytes with phosphate buffer was performed with subsequent derivatization with 9-fluorenylmethyl chloroformate. The quantification was determined by Ultra Performance Liquid Chromatography-tandem mass spectrometer. The method was validated through the following parameters: selectivity, detection and quantification limits, linearity, accuracy, precision and uncertainty. The average recoveries ranged between 94.08 and 103.31%. Additionally, detection limits from 0.396 to 0.433μg/L, and the quantification limit was 5.0μg/L for all the analytes evaluated. In terms of linearity and precision, the results obtained were in the ranges considered adequate (R2 ≥ 0.99 and CV ≤ 20%), the estimated expanded uncertainty was 12.95, 11.15 and 13.83% for glyphosate, aminomethylphosphonic acid and glufosinate, respectively. This method was successfully applied for the determination of the target analytes in irrigation water samples, detecting concentrations of aminomethylphosphonic acid over limit detection for some sampling sites.

Fiber coating based on a green metal-organic framework to determine phthalates in bottled waters by direct-immersion micro solid-phase extraction

The metal-organic framework CIM-80(Al) is proposed as sorbent coating for solid-phase microextraction fibers. The novel CIM-80(Al)-based microextraction fibers (SPME) with a thickness of only 3.5 µm were used to isolate and determine 14 phthalate esters in bottled water with potential results. To compare the extraction efficiency of this novel green material with commercially available SPME fibers, several parameters of the overall procedure were setup. Different parameters such as pH, stirring speed, adsorption and desorption time, salting effect, and percentage of organic solvents were optimized. The results obtained with the CIM-80(Al) based-SPME fibers are promising and comparable to commercial coatings. It demonstrates similar results in a total of 16 bottled water samples subjected to determination. The detection and quantification were performed using ultra-high performance liquid chromatography-tandem mass spectrometer (UHPLC-MS/MS). The effectiveness of the proposed method was corroborated by its analytical characteristics such as limits of detection, which ranged from 0.01 to 0.5 µg L−1 while the linear range was 0.04 to 1 000 µg L−1. In terms of recoveries, CIM-80(Al)-SPME fiber provided results from 70 to 107 %, while the precision of the method in terms of relative standard deviation varied from 1.8 to 18.8 %.

Identification of biomarkers for risk assessment of arsenicosis based on untargeted metabolomics and machine learning algorithms

BackgroundLong-term exposure to inorganic arsenic may lead to arsenicosis. There are, however, currently no validated metabolic biomarkers used for the identification of arsenicosis risk. This study aims to identify metabolites associated with arsenicosis and establish prediction models for risk assessment based on untargeted metabolomics and machine learning algorithms. MethodsIn total, 105 coal-borne arsenicosis patients, with 35 subjects in each of the mild, moderate, and severe subgroups according to their symptom severity, and 60 healthy residents were enrolled from Guizhou, China. Ultra-high performance liquid chromatography-tandem mass spectrometer (UHPLC-MS/MS) was utilized to acquire the plasma metabolic profiles of the studied subjects. Statistical analysis was used to identify disease-associated metabolites. Machine learning algorithms and the identified metabolic biomarkers were resorted to assess the arsenicosis risk. ResultsA total of 143 metabolic biomarkers, with organic acids being the majority, were identified to be closely associated with arsenicosis, and the most involved pathway was glycine, serine, and threonine metabolism. Comparative analysis of metabolites in arsenicosis patients with different symptom severity revealed 422 altered molecules, where disrupted metabolism of beta-alanine and arginine demonstrated the most significance. For risk assessment, the model established by a single biomarker (L-carnosine) could undoubtedly discriminate arsenicosis patients from the healthy. For classifying arsenicosis patients with different severity, the model established using 52 metabolites and linear discriminate analysis (LDA) algorithm yielded an accuracy of 0.970–0.979 on calibration set (n = 132) and 0.818–0.848 on validation set (n = 33). ConclusionAltered metabolites and disrupted pathways are prevalent in arsenicosis patients; The disrupted metabolism of one carbon and dysfunction of antioxidant defense system may partially be causes of the systematic multi-organ damage and carcinogenesis in arsenicosis patients; Metabolic biomarkers, combined with machine learning algorithms, could be efficient for risk assessment and early identification of arsenicosis.

Concentration of osteopontin in human milk and associated factors in Chinese populations from 2011 to 2013

To analyze content of human milk osteopontin(OPN) and to explore associated factors in Chinese populations. The samples and data were extracted from the database for human milk composition in China between 2011 and 2013. A sub-sample of 459 mothers was randomly selected after stratification according to lactation stage, and human milk OPN concentrations were determined by ultra performance liquid chromatography-tandem mass spectrometer(UPLC/MS). The average OPN concentration(M(P25, P75)) in breast milk was 44.0(30.1-72.0) mg/L within 0-330 days postpartum. OPN concentrations were independent of lactation stage, which were 45.6(31.8, 80.7) mg/L in colostrum, 41.3(29.2, 70.0) mg/L in transitional milk and 46.9(30.2, 71.9) mg/L in mature milk, corresponding to 0.40%、0.42% and 0.65% of the total milk protein content(OPN/protein%). The percentage of OPN to total protein in milk showed an increasing trend with lactation progression(r=0.21, P<0.001). Multivariate analysis showed that sleep quality of mothers within one week prior to milk collection was correlated with the breast milk OPN level(P=0.04). The OPN level in breast milk from mothers with good sleep quality was significantly higher than that from mothers with poor sleep quality(46.5 mg/L vs.34.7 mg/L). The median level of milk OPN concentration in mothers from Yunnan was higher than mothers from Beijing(50.5 mg/L vs.36.1 mg/L, P=0.03). Maternal age, mode of delivery, prepregnancy body mass index, weight gain during pregnancy, passive smoking and outdoor activities 24 hours prior to milk collection were not correlated with milk OPN concentration. OPN concentration in breast milk was not related to preterm birth. Also, milk OPN concentration did not correlate with diarrhoea, respiratory disease, or allergic disease in infants during two weeks before milk collection. The concentration of OPN in breast milk of Chinese woman may be similar among different lactation stages. Geographic region and sleep quality of mothers may be related to the milk OPN concentration.

Cocktail Method: Effect of the Bletilla striata Extracts on CytochromeP450 Activity in Rat

Bletilla striata is a dried tuber of B striata (Thunb.) Reichb.f. of Orchidaceae plant, which is mainly used for hemoptysis, vomiting blood, trauma bleeding, sore swollen poison, and cracked skin. There have been few research reports on the effect of this herb on cytochrome P450 (CYP), therefore, the study was aimed to investigate the effects of the B striata extracts on the activity of 6 subtypes (CYP2D6, 1A2, 2C19, 2E1, 3A4, and 2C9) using a cocktail method. The B striata extracts were administrated to rats in 0.21 or 0.63 g/kg once a day for 7 or 14 days. The 3 control groups were used to ensure the accuracy of the results. Subsequently, a cocktail of tolbutamide, chlorzoxazone, midazolam, metoprolol, omeprazole, and caffeine was injected. A ultra performance liquid chromatography–tandem mass spectrometer was developed and validated to investigate the concentration of the probes and the pharmacokinetic parameters were calculated to investigate the effects of the extracts on the activity of 6 enzymes under different doses and different dosing periods. The results suggested that the B striata extracts could induce the activities of CYP2D6, 1A2, and 2C19 and could inhibit the activities of CYP2E1, 3A4, and 2C9. When used in combination with drugs that are metabolized by CYP2D6, 1A2, 2C19, 2E1, 3A4, and 2C9, appropriate dose adjustments were needed to avoid toxic side effects caused by drug interactions.

Anti-Inflammatory Potentials of the n-Hexane Fraction of Alstonia boonei Stem Bark in Lipopolysaccharide-Induced Inflammation in Wistar Rats.

BackgroundInflammation is a protective response of the host to infections and tissue damage and medicinal plants have been used to regulate inflammatory response. The phytochemical contents of the n-hexane fraction of Alstonia boonei and their anti-inflammatory potentials in lipopolysaccharide-induced inflammation were investigated in rat liver.Materials and MethodsA quantity of 5 mg/kg lipopolysaccharide (LPS) was used to induce inflammation in twenty-five male Wistar rats, grouped (n = 5) and treated as follows: negative control (10 mL/kg saline), positive control (1 mg/kg ibuprofen); 50, 100 and 20 mg/kg of the n-hexane fraction of Alstonia boonei were administered to test groups. In another experiment, twenty rats (n = 5, without LPS) were administered the same doses of the n-hexane fraction of A. boonei and ibuprofen for seven days. At the end of the experiment, animals were sacrificed, serum was obtained from blood and liver mitochondria isolated in a refrigerated centrifuge. Mitochondrial permeability transition (mPT) pore opening and mitochondrial F0F1 ATPase (mATPase) were determined spectrophotometrically. Serum interleukins 1β, 6 (IL-1β, IL-6), tumour necrosis factor alpha (TNF-α), C-reactive protein (CRP) and creatine kinase (CK), gamma glutamyl transferase (GGT), aspartate and alanine aminotransferases (AST and ALT,) of the animals in which inflammation was induced using LPS but treated with graded doses of n-hexane fraction of A. boonei were determined using the ELISA technique. The phytochemical contents of the n-hexane fraction of A. boonei were determined using ultra performance liquid chromatography-tandem mass spectrometer (UHPLC-MS).ResultsCalcium induced mPT in 8 fold and LPS induced mPT 14 fold in the negative control while the n-hexane fraction reversed mPT in the treated groups (50, 100 and 200 mg/kg) to 2, 4, 4 folds, respectively. LPS treatment of the negative group enhanced F0F1 mATPase activity, increased CRP, TNF-α, IL-1β, IL-6 levels as well as CK, AST, ALT and GGT activities. These values were significantly reduced by 100 and 200 mg/kg of the n-hexane fraction. UHPLC-MS analysis of the fraction revealed the presence of terpenoids, phenolics and sphingolipids.ConclusionThese results showed that bioactive phytochemicals present in the n-hexane fraction of A. boonei were not toxic, have an anti-inflammatory effect and could be used for the treatment of inflammatory diseases.

Study on the performance of vanadium doped NaNbO3 photocatalyst degradation antibiotics

In this paper, by adjusting the mass ratio of Nb2O5, NaOH and V2O5, the hydrothermal synthesis method was used to successfully dope vanadium into NaNbO3 to prepare Vanadium-doped NaNbO3 powder. The modified constitution and structure of Vanadium-doped NaNbO3 are characterized through X-ray Diffraction, Scanning Electron Microscope and Energy Spectrum, and Ultraviolet–Visible Diffuse Reflection Spectrum representation. The results show that doping with V2O5 can reduce the band gap of NaNbO3 and increase its utilization of visible light. Under light radiation, Vanadium-doped NaNbO3 exhibits excellent photocatalytic degradation activity for the degradation of tetracycline, ciprofloxacin and enrofloxacin. The degradation rate of modified NaNbO3 with the most applicable quantity of V2O5 on Tetracycline within 90 min can reach 61%, the degradation rate on Ciprofloxacin within 150 min can reach 76%, and the degradation rate on Enrofloxacin within 150 min can reach 64.6%. Through cycled degradation experiment, we find that Vanadium-doped NaNbO3 Photocatalyst has good photocatalytic stability. This paper also uses high performance liquid chromatography-tandem mass spectrometer to analyze the tetracycline solution under different light time, and speculates the molecular formula and structural formula of the intermediate product that may be produced by the tetracycline molecule in the photocatalytic degradation process. In addition, the photocatalytic degradation mechanism and possible degradation pathways of tetracycline were also discussed. This article can provide a reference for the actual antibiotic wastewater treatment.

Effects of Herba Erigerontis injection on pharmacodynamics and pharmacokinetics of warfarin in rats in vivo.

Herba Erigerontis injection (HEI) is an aqueous solution derived from whole plants of Erigeron breviscapus, which may be co-administered with warfarin to treat cardiovascular and cerebrovascular disorders. This research was conducted to make sure whether HEI would affect anticoagulation of warfarin to guarantee reasonable medication. The pharmacodynamic study was designed to measure prothrombin time (PT) and activated partial thromboplastin time (APTT) values, and international normalized ratio (INR) values were calculated. For pharmacokinetic study, ultra performance liquid chromatography-tandem mass spectrometer (UPLC-MS/MS) technology was applied to measure plasma concentrations of warfarin enantiomers. The influence of HEI on plasma protein binding rate of warfarin was assessed by ultrafiltration. Pharmacodynamic study demonstrated that both HEI alone and co-administered with warfarin could increase PT and INR values significantly (P<.01), whereas the APTT values were unaffected (P>.05). Pharmacokinetic study manifested that Cmax , AUC and t1/2 prolonged significantly (P<.01) for R/S-warfarin in presence of HEI. Low (3.6mL/kg), medium (7.2mL/kg) and high (10.8mL/kg) doses of HEI could decrease plasma protein binding rate of warfarin significantly (P<.01). The results mean that HEI can potentiate the anticoagulant response of warfarin through both pharmacodynamics and pharmacokinetics.

Anthocyanins in Blueberries Grown in Hot Climate Exert Strong Antioxidant Activity and May Be Effective against Urinary Tract Bacteria

Anthocyanins are extensively studied for their health-related properties, including antibacterial activity against urinary tract infections (UTI). Among common fruits, blueberries, with their remarkable antioxidant capacity, are one of the richest sources. Anthocyanin-rich extracts were obtained from four varieties: Snowchaser, Star, Stella Blue and Cristina Blue, grown in the hot climate of Southern Spain. Their total anthocyanins contents (TAC) were determined spectrophotometrically, and the anthocyanin profile by ultra high performance liquid chromatography—tandem mass spectrometer (UHPLC-MS/MS). Their antioxidant activity was assessed by oxygen radical absorbance capacity (ORAC) assay, while antibacterial activity against strains isolated from UTI patients was assessed in vitro, helping to select the varieties with the highest bioactive potential. Star showed the highest TAC and antioxidant activity (1663 ± 159 mg of cyanidin-3-O-glucoside (cy-3-O-glu) equivalents/100 g fresh weight (FW), 6345 ± 601 μmol Trolox equivalents (TE)/100 g FW, respectively), followed by Cristina Blue, Stella Blue and Snowchaser. As far as we know, this is the first time that cyanidin-3-rutinoside has been identified in blueberries. The extracts inhibited all the tested strains, MICs ranging from 0.4 mg/mL (for Stella Blue extract against UTI P. aeruginosa) to 9.5 mg/mL (for all extracts against UTI K. pneumoniae ssp. pneumoniae). This is the first study that assessed in vitro the antibacterial activity of blueberries against Klebsiella pneumoniae, Providencia stuartii and Micrococcus spp. strains isolated from UTI.

Safety and Activity of the Combination of Ceritinib and Dasatinib in Osteosarcoma.

Osteosarcoma (OS) is the second most common cause of cancer-related death in pediatric patients. The insulin-like growth factor (IGF) pathway plays a relevant role in the biology of OS but no IGF targeted therapies have been successful as monotherapy so far. Here, we tested the effect of three IGF specific inhibitors and tested ceritinib as an off-target inhibitor, alone or in combination with dasatinib, on the proliferation of seven primary OS cells. Picropodophyllin, particularly in combination with dasatinib and the combination ceritinib/dasatinib were effective in abrogating the proliferation. The ceritinib/dasatinib combination was applied to the primary cells of a 16-year-old girl with a long history of lung metastases, and was more effective than cabozantinib and olaparib. Therefore, the combination was used to treat the patient. The treatment was well tolerated, with toxicity limited to skin rush and diarrhea. A histopathological evaluation of the tumor after three months of therapy indicated regions of high necrosis and extensive infiltration of macrophages. The extension of the necrosis was proportional to the concentration of dasatinib and ceritinib in the area, as analysed by an ultra performance liquid chromatography–tandem mass spectrometer (UPLC-MS/MS). After the cessation of the therapy, radiological analysis indicated a massive growth of the patient’s liver metastases. In conclusion, these data indicate that the combination of ceritinib/dasatinib is safe and may be used to develop new therapy protocols.

A Novel Eye Drop Candidate for Age-Related Macular Degeneration Treatment: Studies on its Pharmacokinetics and Distribution in Rats and Rabbits.

Age-related macular degeneration (AMD) is wearing down of macula of retina, causing a blur or loss of vision in the center of the visual field. It can be categorized into dry or wet AMD. Until now, medical treatments for dry AMD have not been developed yet. The aim of this study was to evaluate pharmacokinetics (PKs) and tissue distribution of CK41016, a novel candidate for dry AMD, after intravenous (IV) or eye drop administration in rats and rabbits. In addition, a simple and sensitive bioanalytical method for CK41016 using ultra performance liquid chromatography-tandem mass spectrometer (UPLC-MS/MS) was developed. PK parameters were estimated by compartmental analysis using a WinNonlin® software version 8.1 (a Certara™ company). A PK model of CK41016 was well-described by the two-compartment model. The tissue-to-plasma partition coefficient (Kp) of CK41016 was the highest in the vitreous humor of rats and the cornea of rabbits after eye drop administration. In addition, the Caco-2 cell transporter assay confirmed that CK41016 was not an active substrate for the efflux transporter. In summary, the PKs and tissue distribution of CK41016 were successfully evaluated and investigated whether this drug was a substrate of efflux transporters.

UPLC-MS/MS study of the effect of dandelion root extract on the plasma levels of the selected irreversible tyrosine kinase inhibitors dasatinib, imatinib and nilotinib in rats: Potential risk of pharmacokinetic interactions.

Tyrosine kinase inhibitor treatments for chronic myeloid leukaemia based on nilotinib (NIL), dasatinib (DAS) and imatinib (IMA) have improved patient quality of life and have turned chronic myeloid leukemia from a fatal disease into a chronic disease. Dandelion is a rich source of phenolic compounds with strong biological properties, and the effects of using this plant in the treatment of different illnesses can be linked to the presence of various polyphenols found in the different parts of the plant. Thus, dandelion can potentially be used as a nutraceutical (dietary antioxidant) to prevent different disorders associated with oxidative stress, i.e. cardiovascular disorders, cancer and inflammatory processes. Mutual interference between a drug and a food constituent may result in altered pharmacokinetics of the drug and undesired or even dangerous clinical situations. In the present study, a bioanalytical ultra performance liquid chromatography-tandem mass spectrometer (UPLC-MS/MS) method was developed and validated for the quantification of DAS, IMA and NIL in rat plasma. Sample preparation was carried out using solid-phase extraction with C18 cartridges with a good extraction recovery of ≥94.37% for the three drugs. The method was fully validated as per the US Food and Drug Administration guidelines.

Development and Validation of the Ultra Performance Liquid Chromatography-Tandem Mass Spectrometer Method for Quantification of Methylenecyclopropylglycine in Litchi Fruits Using the Standard Addition Method

Recently, the presence of methylenecyclopropylglycine (MCPG) found in litchi seeds and ripe and unripe litchi fruit pulp proved to be a breakthrough finding as it causes hypoglycemic encephalopathy leading to death of under nourished/starved children. Earlier methods of isolation and detection of MCPG in litchi fruit were tedious and time-consuming. To reduce the complexity, we developed a better and straightforward methodology using ultra performance liquid chromatography-tandem mass spectrometer (UPLC-MS/MS) and QuEChERS technique to quantify MCPG without derivatization in litchi samples. The UPLC system included a Thermo C8 column with a mobile phase 0.1% formic acid in acetonitrile and water (gradient flow) at a flow rate of 0.3 ml/min. MCPG levels were determined using an API 4000 mass spectrometer with electrospray ionization (ESI) source. Other parameters were also determined following the quantitation by multiple reaction monitoring mode. The method was validated for selectivity, specificity, linearity, limit of quantification (LOQ), limit of detection (LOD), intra-inter day precision and accuracy, stability, and ruggedness. Calibration curve ranged from 7.8 to1000 ng/ml linearly while the LOD value for MCPG was 0.66 ng/ml. The percent accuracy and precision ranged from 92.98 to 105% and 1.78 to 8.43%, respectively. Validated method was applied for the determination of MCPG in litchi samples which provided a better and novel method for the determination of MCPG.

Human urine contamination with environmental pollutants: simultaneous determination using UPLC-MS/MS.

Paraben derivatives are widely used as an antifungal, antimicrobial preservative in cosmetic products, pharmaceuticals, and food. These molecules are called endocrine disruptors (EDCs). The exposure of the human body to paraben derivatives needs further study and for this purpose 200 urine samples were collected from Tunisian men and women aged between 5 and 90 years to determine three paraben derivatives: methylparaben (MP), ethylparaben (EP) and propylparaben (PP) using ultra performance liquid chromatography-tandem mass spectrometer (UPLC-MS/MS). The three major parabens were found in 95 urine samples. The obtained results indicate that MP, EP, and PP were detected in 57%, 46%, and 40% of all samples, respectively. Urinary concentration for the three parabens was in the range of 0.88-84.46 ng/mL, 0.52-29.2 ng/mL, and 0.51-28.17 ng/mL of PP, MP, and EP, respectively. In addition, the concentrations of the paraben derivatives in women were higher than those of men. These findings indicate that the exposure occurs from common products (foods, cosmetics, and pharmaceuticals). The Tunisian authorities should control the composition of packaging of these common products in order to protect humans against EDCs.

Application of synthesized nano-CuS photocatalyst for degradation of Ofloxacin and its by-products

ABSTRACTIn the present work, the synthesis, characterization, and photocatalytic performance of CuS nanopowder were studied. The achieved photocatalyst was characterized by X-ray diffraction, Scanning Electron Microscopy, X-ray photoelectron spectroscopy, and Brunauer–Emmett–Teller. The degrading characteristic of CuS photocatalyst was investigated during 100 min and 18 h under visible radiation (Vis irradiation) for degradation of Ofloxacin (OFX), revealing a good degradation rate of OFX. Over 100 min of degradation, some by-products that can be considered as secondary pollutants were produced. Then, to destroy these pollutants the radiation time was increased up to 18 h. The results showed a perfect degradation of OFX and its secondary pollutants. The effective variables including pH, degradation time, and photocatalyst dosage were optimized and studied in multivariate method by design expert7 software. Moreover, the kinetic of the process was evaluated, showing accordance with Langmuir–Hinshelwood model. Recognition of the products was carried out by ultra performance liquid chromatography-tandem mass spectrometer. The synergic effect of CuS adsorbent-photocatalyst exhibited a good adsorption-degradation rate of OFX under Vis irradiation.

Determination and distribution of pesticides and antibiotics in agricultural soils from northern China.

Different types of soil samples from a typical farmland in northern China were collected and evaluated for the presence of the pesticides and antibiotics. 47 pesticides were extracted with a quick, easy, cheap, effective, rugged, and safe (QuEChERS) preparation method and cleanup with 50 mg C18, while 10 antibiotics were extracted with methanol/EDTA–McIlvaine buffer solution (v/v = 1/1), then both of them were analyzed with high performance liquid chromatography-tandem mass spectrometer (HPLC-MS/MS). Total concentrations of the 47 pesticides in the soil samples ranged from not detectable (ND) to 3.8 mg kg−1. The soil exhibited relatively high ecological risk for atrazine, chlorpyrifos, tebuconazole, difenoconazole, pymetrozine, and thiamethoxam, as over 1.0% of the sample concentrations exceeded 0.1 mg kg−1. The residual levels of the 10 antibiotics were relatively low (ND-951.0 μg kg−1). Tetracyclines exhibited a high detection rate (20.9%), with 2.8% of the soil samples exhibiting tetracyclines concentrations exceeding 100 μg kg−1, implying high ecological risk. The 4 sulfonamides and 2 macrolides analyzed showed detection rates below 0.8%. Spatial changes in the distribution of pesticides and antibiotics appear to be related to land use patterns, particularly orchards and vegetable plots. The over-standard rate of pesticides and antibiotics in orchards was greater than that of vegetable plots, and grain fields had the lowest over-standard rate. These data were helpful to figure out the pollution of these pesticides and antibiotics, and provided valuable information for soil quality assessment and risk assessment.

Electrochemical simulation of three novel cardiovascular drugs phase I metabolism and development of a new method for determination of them by liquid chromatography coupled with tandem mass spectrometry

In this study electrochemistry (EC) coupled with electrospray ionization mass spectrometry (ESI-MS) was used to study the metabolic fate of three novel cardiovascular drugs: rivaroxaban (RIV), aliskiren (ALS), and prasugrel (PRS). Mimicry of the oxidative phase I metabolism was achieved in a simple amperometric thin-layer cell equipped with a boron-doped diamond (MD) working electrode. Structures of the electrochemically-generated metabolites were elucidated from MS/MS experiments. Additionally, a sensitive, specific, and rapid ultra-high performance liquid chromatography–tandem mass spectrometer (UHPLC–MS/MS) method has been developed and validated for the selected drugs in human urine samples. Three different sample preparation methods were compared and finally, sample preparation was accomplished through an ultrasound-assisted emulsification microextraction process (USAEME). The drugs were detected using a triple quadrupole tandem mass spectrometer by multiple reaction monitoring via an electrospray ionization source with positive ionization mode (ESI(+)). The results obtained by EC–MS were compared with conventional in vivo studies by analyzing urine samples from patients. Data from in vivo experiments showed good agreement with the data from electrochemical oxidation. Thus, EC–MS is very well-suited for the simulation of the oxidative metabolism of rivaroxaban, aliskiren, and prasugrel as well. Moreover, electrochemical conversion of target compounds appears to be a new in vitro technology for the prediction of potential metabolites.

Ocular Pharmacokinetics of 25-Hydroxyvitamin D3 After Weekly Supplementation in Rabbits Using Ultra Performance Liquid Chromatography-Tandem Mass Spectrometer.

The protective role of vitamin D supplementation has recently been shown to be present in various ocular inflammatory diseases. The oral supplementation of vitamin D may take time to achieve adequate levels in intraocular fluids. Therefore, the present study was performed to understand the ocular pharmacokinetics of 25-hydroxyvitamin D3 (25D3) in aqueous humor after weekly supplementation of 25D3 in rabbits. A total of 21 rabbits were fed orally with 25D3 (7.22µg/kg/week) for 8weeks and 9th dose was given at the end of 8weeks. The blood and aqueous humor samples were collected from ear vein and though anterior chamber paracentesis, respectively. The serum and aqueous humor samples were spiked with deuterium labeled internal standard and were extracted using liquid extraction method. Furthermore, the samples were derivatized and 25D3 estimation was performed using ultra performance liquid chromatography-tandem mass spectrometer (UHPLC-MS/MS). The 25D3 supplementation significantly increased the 25D3 levels in serum (78.5 ± 21.6ng/ml) (mean ± SD) (p < 0.0001) and in aqueous humor (991.3 ± 180.6pg/ml) (mean ± SD) (p < 0.0001) compared to baseline levels. The maximum concentration was achieved in serum after the 10th hour of supplementation of 1st and 9th dose, while the same was observed at the 24th hour in aqueous humor. The oral supplementation of 25D3 was found to significantly increase 25D3 levels in aqueous humor; however, the time required to achieve 25D3 concentration in aqueous humor was higher as compared to that in serum. Therefore, weekly oral supplementation of 25D3 may have a beneficial role in ocular diseases.

Bio-based synthesis of silver nanoparticles from orange waste: effects of distinct biomolecule coatings on size, morphology, and antimicrobial activity.

PurposeDespite the numerous reports on biological syntheses of silver nanoparticles (AgNPs), little is known about the composition of their capping agents, protein corona of plant extract-mediated synthesis, and their influence on the properties of AgNPs. Here, orange (Citrus sinensis) waste was utilized as a source of an extract for AgNP synthesis (the protein corona composition of which was elucidated), and also as a starting material for hesperidin and nanocellulose extraction, which were used for bio-based AgNP synthesis. A comparison of the results using the two methods of synthesis is presented.MethodsAgNPs were synthesized using orange (C. sinensis) peel extract (Or-AgNPs) in a biological route, and using hesperidin (Hsd-AgNPs) and nanocellulose (extracted from oranges) in a green chemical route. Characterization of nanoparticles was carried out using zeta potential and hydrodynamic size measurements, transmission electron microscopy, and X-ray diffraction. Elucidation of proteins from protein corona was performed via ultra performance liquid chromatography-tandem mass spectrometer experiments. Antimicrobial activity was assessed via minimum inhibitory concentration assays against Xanthomonas axonopodis pv. citri (Xac), the bacterium that causes citric canker in oranges.ResultsOr-AgNPs were not completely uniform in morphology, having a size of 48.1±20.5 nm and a zeta potential of −19.0±0.4 mV. Stabilization was performed mainly by three proteins, which were identified by tandem mass spectrometry (MS/MS) experiments. Hsd-AgNPs were smaller (25.4±12.5 nm) and had uniform morphology. Nanocellulose provided a strong steric and electrostatic (−28.2±1.0 mV) stabilization to the nanoparticles. Both AgNPs presented roughly the same activity against Xac, with the minimum inhibitory concentration range between 22 and 24 μg mL−1.ConclusionDespite the fact that different capping biomolecules on AgNPs had an influence on morphology, size, and stability of AgNPs, the antibacterial activity against Xac was not sensitive to this parameter. Moreover, three proteins from the protein corona of Or-AgNPs were identified.

An LC–MS/MS method for simultaneous determination of nine steroidal saponins from Paris polyphylla var. in rat plasma and its application to pharmacokinetic study

Paris polyphylla var is an herbal plant herb widely used in Traditional Chinese Medicine. The purpose of this study is to develop an Ultra Performance Liquid Chromatography-tandem mass spectrometer (UPLC–MS) method to quantify the major components (i.e., nine saponins) from P. polyphylla in plasma samples. A UItra BiPh column (100×2.1mm, 5μm) was used with acetonitrile/0.1% formic acid in water as mobile phases. The analytes were quantified using a Waters XEVO TQ mass spectrometer via multiple reaction monitoring (MRM) with positive scan mode. A protein precipitation method was used to extract the analytes from rat plasma. The inter/intra-day precision, accuracy, recovery, matrix effect, and stability were evaluated per the FDA guidance. The method showed linearity in the concentration ranges of 2.4–1250ng/mL. The intra-day and inter-day precisions (RSD) of these analytes at three different levels were less than 15.0%. The extraction recoveries of these analytes were from 83.8% to 109.4% and the matrix effects ranged from 87.4% to 105.4%. The stabilities of these compounds in plasma were evaluated by analyzing three different concentrations following storage at 25°C for 6h, and −80°C for 30days. All the samples displayed less than 15.0% variations. The validated method was successfully used to a pharmacokinetic (PK) study using Sprague Dawley (SD) rats with intravenous (i.v.) and oral (p.o.) administration of P. polyphylla extract. The applications revealed that this method can be used to analyze major steroidal saponins from P. polyphylla in biological samples.