Comparative evaluation of SPE methods for biotoxicity assessment of water and wastewater: Linkage between chemical extracting efficiency and biotoxicity outcome.

Biotoxicity assessment results of environmental waters largely depend on the sample extraction protocols that enrich pollutants to meet the effect-trigger thresholds of bioassays. However, more chemical mixture does not necessarily translate to higher combined biotoxicity. Thus, there is a need to establish the link between chemical extracting efficiency and biotoxicity outcome to standardize extraction methods for biotoxicity assessment of environmental waters. This study compares the performance of five different extraction phases in solid phase extraction (SPE), namely HLB, HLB+Coconut, C18 cartridge, C18 disk and Strata-X, and evaluated their chemical extracting efficiencies and biotoxicity outcomes. We quantitatively assessed cytotoxicity, acute toxicity, genotoxicity, estrogenic activity, and neurotoxicity of the extracts using in vitro bioassays and characterized the chemical extracting efficiencies of the SPE methods through chemical recoveries of 23 model compounds with different polarities and total organic carbon. Using Pareto ranking, we identified HLB+Coconut as the optimal SPE method, which exhibited the highest level of water sample biotoxicity and recovered the most chemicals in water samples. We found that the biotoxicity outcomes of the extracted water samples significantly and positively correlated with the chemical extracting efficiencies of the SPE methods. Moreover, we observed synchronous changing patterns in biotoxicity outcome and chemical extracting efficiencies in response to increasing sample volumes per cartridge (SVPC) during SPE. Our findings underscore that higher chemical extracting efficiency of SPE corresponds to higher biotoxicity outcome of environmental water samples, providing a scientific basis for standardization of SPE methods for adequate assessment of biotoxicities of environmental waters.

Bioavailability assessment of a brompheniramine taste-masked pediatric formulation in a juvenile porcine model.

A brompheniramine taste-masked pediatric formulation was developed as part of the National Institutes of Health Pediatric Formulation Initiative to help address low patient compliance caused by the bitter taste of many adult formulations. To confirm that the taste-masked formulation can provide a similar pharmacological effect to the previous marketed adult formulations, a juvenile porcine model was used to screen the model pediatric formulation to compare the bioavailability between the marketed brompheniramine maleate and the taste-masked maleate/tannate formulation. Pigs were dosed orally with both formulations and blood samples were obtained from 0 to 48 h. Plasma samples were prepared and extracted using solid-phase extraction. The mass spectrometer was operated under selected ion monitoring mode. The selected ion monitoring channels were set to m/z 319.1 for brompheniramine and m/z 275.2 for the internal standard chlorpheniramine. Calibration curves were linear over the analytical range 0.2-20 ng/ml (r2 > 0.995) for brompheniramine in plasma. The intra- and inter-day accuracies were between 98.0 and 105% with 5.73% RSD precision. The bioanalytical method was successfully applied to a preclinical bioavailability study. The bioavailability profiles were not significantly different between the two formulations, which demonstrates that taste-masking with tannic acid is a promising approach for formulation modification for pediatric patients.

Method development of multi pesticide residue analysis in country beans collected from Dhaka, Bangladesh, and their dietary risk assessment.

The aim of the study was to develop a modified QuEChERS method coupled with liquid chromatography-tandem mass spectrometry (LC-MS/MS) for the simultaneous determination of five multi-class pesticides in country beans collected from Dhaka, Bangladesh. Pesticides were extracted using ACN, and to minimize the co-extraction matrix, optimized d-SPE cleanup was done using sorbents (GCB, PSA, and C18). In the calibration range, the method showed excellent linearity with a correlation coefficient of R2 ≥ 0.9990 both in solvent- and matrix-matched calibration. For the selected pesticides, average recoveries (at four spiking levels (n = 5) of 10, 20, 100, and 200 µg/kg) of 70-100 % were achieved with relative standard deviations (RSDs) ≤ 9.5 %. The limit of detection (LOD) and limit of quantification (LOQ) ranged from 0.3333 to 1.3333 µg/kg and 1.0 to 4.0 µg/kg, respectively. The dietary risk assessment, in terms of hazard quotient (HQ), was calculated to assess consumers' health risks.

Less is more: a methodological assessment of extraction techniques for per- and polyfluoroalkyl substances (PFAS) analysis in mammalian tissues.

Per- and polyfluoroalkyl substances (PFAS) are persistent environmental contaminants. Studying the bioaccumulation in mammalian tissues requires a considerable effort for the PFAS extraction from complex biological matrices. The aim of the current work was to select and optimize the most efficient among common extraction strategies for eleven perfluoroalkyl acids (PFAA). Primary extractions from wild boar tissues (liver, kidney, and lung) were performed with methanol at neutral, acidic, or alkaline conditions, or with methyl-tert-butyl ether (MTBE) after ion-pairing with tetrabutylammonium (TBA) ions. A second purification step was chosen after comparing different solid-phase extraction (SPE) cartridges (Oasis WAX, ENVI-Carb, HybridSPE Phospholipid) and various combinations thereof or dispersive SPE with C18 and ENVI-Carb material. The best extraction efficiencies of the liquid PFAA extraction from tissue homogenates were achieved with methanol alone (recoveries from liver 86.6-114.4%). Further purification of the methanolic extracts using dispersive SPE or Oasis WAX columns decreased recoveries of most PFAA, whereas using pairs of two SPE columns connected in series proved to be more efficient albeit laborious. Highest recoveries for ten out of eleven PFAA were achieved using ENVI-Carb columns (80.3-110.6%). In summary, the simplest extraction methods using methanol and ENVI-Carb columns were also the most efficient. The technique was validated and applied in a proof of principle analysis in human tissue samples.

Multiclass determination of drug residues in water and fish for bioaccumulation potential assessment.

In this work, a wide-scope liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for the quantitative determination of environmental levels of multiclass drugs and their metabolites in water and fish samples was developed. The method allowed the reliable determination of 44 drugs, covering a rather wide range of chemistries and physicochemical characteristics. In order to obtain a reliable and robust analytical protocol, different combinations of extraction and cleanup techniques were systematically examined. Aqueous samples were extracted using a simple Oasis HLB SPE enrichment protocol with pH-optimized sample percolation (pH 3). The extraction of cryo-homogenized biota samples was performed using double extraction with MeOH basified with 0.5% NH3, which allowed high extraction recoveries for all target analytes. The problem of the coextracted lipid matrix, which is known to be the key obstacle for reliable biota analysis, was systematically examined in a series of model cleanup experiments. A combination of cryo-precipitation, filtration, and HLB SPE cleanup was proposed as a protocol, which allowed reliable and robust analysis of all target compounds at low ng/g levels. At the final conditions, the method which was validated at three concentration levels showed high extraction recoveries (68-97%), acceptable matrix effects (12 to -32%), accuracies (81-129%), and reproducibilities (3-32%) for all analytes. The developed method was used to determine drug concentrations in river water and in feral freshwater fish, including whole fish and muscle tissue, from the Sava River (Croatia), in order to estimate their corresponding bioaccumulation potential. With respect to bioaccumulation potential in whole fish and fish muscle, the most relevant drugs were lisinopril, sertraline, terbinafine, torsemide, diazepam, desloratadine, and loratadine with estimated bioaccumulation factors ranging from 20 to 838 and from 1 to 431, respectively.

Determination of benzothiazoles, benzotriazoles and benzenesulfonamides in seafood using quick, easy, cheap, effective, rugged and safe extraction followed by gas chromatography - tandem mass spectrometry: Method development and risk assessment.

The common use of benzothiazoles, benzotriazoles and benzenesulfonamides has led to widespread ubiquity in several environmental matrices. Their occurrence in edible fish could represent an additional exposure route for the population. The present study aims to develop a method for the simultaneous determination of these three compound families in seafood samples. Based on QuEChERS extraction, different salt combinations and clean-up strategies have been evaluated to achieve the highest recoveries while reducing the matrix effect in low and high lipidic content species. The best results were obtained with the original method salts and the lipid-selective push-through clean-up, which combined with gas chromatography-tandem mass spectrometry led to recoveries between 50 and 112% with negligible matrix effects and method detection limits between 0.15-9.50 ng g-1 dw. The application of the method to commercially available samples confirmed the presence of BTs as well as BSAs, with the latter being determined in seafood for the first time. Exposure and risk assessment calculations indicated a minor risk for the population when consuming fish.

Highly sensitive detection of okadaic acid in seawater by magnetic solid-phase extraction based on low-cost metal/nitrogen-doped carbon nanotubes.

Algae toxins pose a severe threat to human health all over the world. In this study, magnetic metal/nitrogen-doped carbon nanotubes (M-NCNTs) were facilely synthesized based on one-step carbonization and applied for magnetic solid-phase extraction of okadaic acid (OA) from seawater followed by high performance liquid chromatographic tandem mass spectrometry (HPLC-MS/MS) analyses. Differences in the physicochemical properties of the three prepared materials (Fe/Co/Ni-NCNTs) were investigated to confirm the best extraction material. Among them, Ni-NCNTs demonstrated a faster extraction rate (10 min) and higher adsorption capacity (223.5 mg g-1), mainly due to the higher specific surface area, suitable pore structure and more abundant pyridine nitrogen ring. Under the optimal conditions, the calibration curve was linear over the range (1.0-800.0 pg mL-1) with good determination coefficients (R) of 0.9992. The limit of detection (LOD) obtained in multiple replicates was 0.4 pg mL-1. Three seawater samples were measured by the developed method, 12.3 pg mL-1 of OA was detected with a satisfying recovery (88.6%-106.7%) and acceptable repeatability (RSD ≤ 4.8%, n = 6). The results demonstrate that M-NCNTs materials are a promising candidate for magnetic solid-phase extraction. Benefiting from its high extraction and interference resistance, the established analytical method is expected to be extended to detect other marine environmental pollutions.

A low-cost eco-friendly fast drug extraction (FaDEx) technique for environmental and bio-monitoring of psychoactive drug in urban water and sports-persons' urine samples.

Nicotine is the most prominent psychoactive/addictive chemical substance consumed worldwide among young players in team sports. Moreover, urinary nicotine discharge and nicotine-based products disposal in environmental waters has been unavoidable in recent years. Therefore, sensitive monitoring of nicotine content in environmental waters and human urine samples is essential. In this study, we developed a miniaturized novel green, low-cost, sensitive, in-syringe-based semi-automated fast drug extraction (FaDEx) protocol coupled with gas chromatography-flame ionization detection (GC-FID) for the efficient environmental and bio-monitoring of nicotine in aqueous samples. The FaDEx method consists of two steps; firstly, the target analyte was extracted using dimethyl carbonate (a green solvent) and extraction salts. After that, the extraction solvent was passed automatically through the solid-phase extraction cartridge at a constant flow rate for the cleanup process to achieve the sensitive nicotine analysis by GC-FID. Under optimized experimental conditions, the developed method showed excellent linearity over the concentration ranges between 20-2000 ng mL-1 with a correlation coefficient >0.99. The detection and quantification limits were 4 and 20 ng mL-1, respectively. The presented method was applied to monitor and assess nicotine exposure in sports-persons' urine and environmental water samples. The method accuracy and precision in terms of relative recovery and relative standard deviation (for triplicate analysis) were 85.4-110.2% and ≤8%, respectively. Finally, the impact of our procedure on the environment from a green analytical chemistry view was assessed using a novel metric system called AGREE, and obtained the greenness score of 0.87, indicating its an efficient alternative green analytical protocol for routine environmental and bio-monitoring of nicotine in environmental and biological samples.

Multiresidue analysis and probabilistic dietary risk assessment of 241 pesticides in wheatgrass (Triticum sp.) using LC-MS/MS in combination with QuEChERS extraction.

Wheatgrass is consumed as an important nutritious herbal food supplement across the globe; however, limited studies have been reported analyzing multiclass pesticides in this complex, nutrient-rich natural product. An analytical method was developed for the estimation of 241 pesticides in random wheatgrass samples collected from Delhi Northern Capital Region (Delhi-NCR). Extraction was performed by QuEChERS, cleaning was performed by dispersive solid-phase extraction and the extracts were analyzed using triple quadrupole liquid chromatography mass spectrometry. The limit of quantification was 0.5 µg/kg, which is well below the European Union Maximum Residue Level. The coefficient of determination was >0.991 across a calibration range of 0.5-100 µg/kg. The relative standard deviation values for 231 pesticides based on 10 replicates of samples spiked at 10 µg/kg were <5%. Among random samples, 54% confirmed the presence of at least one pesticide. The results indicated the presence of eight different pesticides among 38% of the total population with metribuzin at 299.7 µg/kg and carfentrazone-ethyl at 19.47 µg/kg exceeding the permissible limits among 6% of the total estimated population. The chronic and acute risk quotients as calculated were <1, indicating nonsignificant dietary risk to consumers. However, the presence of pesticides above the permissible limit is likely to result in adverse health effects to the consumers of herbal supplements from an urban population and incorporating measures would be useful to ensure the quality and safety of wheatgrass consumption.

Suspected-screening assessment of the occurrence of organic compounds in sewage sludge.

The profiling of emerging organic pollutants present in sludge and generated during wastewater treatment is much more limited than in water. This is mainly due to the difficulty of sludge analysis because of its high content of organic matter and interfering compounds. In this study, a generic extraction method using a mixture of buffered water (pH 4.1) and solid phase extraction (SPE) clean-up was applied to samples of sludge obtained in different treatment plants. This extraction was followed by determination of the contaminants by ultra-high performance liquid chromatography coupled to high resolution mass spectrometry (UHPLC-HRMS), using suspected screening to detect the most relevant organic compounds that access the environment through sludge application. This screening (including >3000 substances, such as, pharmaceuticals, pesticides, metabolites and industrial chemicals) tentatively identified 122 compound and assigned most probable structure to 39. The set of compounds assigned to a probable structure was increased in 14 compounds by searching in a free database of metabolites. Fifteen compounds were unequivocally confirmed against the analytical standard. Pharmaceuticals and personal care products (PPCPs), with 31 substances identified and 8 confirmed were the main group of compounds. Compounds frequently detected in all sludge samples include nucleotides such as adenosine triphosphate, amino acids such as phenylalanine, or peptides such as leu-phe. Altogether, the results of this work highlight the interest of HRMS to draw the profile of organic compounds in complex matrices.

Waste toner-derived micro-materials as low-cost magnetic solid-phase extraction adsorbent for the analysis of trace Pb in environmental and biological samples.

Lead (Pb) is a toxic heavy metal and is commonly used in industrial applications. Thus, Pb poisoning is a concerning public health issue worldwide. The amounts of lead in natural water, urine, and blood can serve as significant indicators for monitoring the exposure of Pb poisoning. Waste toner has the characteristics of both "waste" and "resource," as it is a "resource in the wrong place." Here, a low-cost carboxylate-functionalized magnetic adsorbent was first synthesized from waste toner by a simple thermal treatment and served as a novel adsorbent with a flexible multidentate O-donor for pre-concentration of trace Pb. The characterization, adsorption behavior, and various factors of adsorption and desorption were adequately optimized, and prior to graphite furnace atomic absorption spectrometry (GFAAS) detection, a new magnetic solid-phase extraction method was proposed for the analysis of Pb in real environmental water and biological samples. The developed method exhibited a low detection limit (0.003 µg L-1), high enrichment factor (88.6-fold), good linearity (0.01-0.3 µg L-1), satisfactory precision with relative standard deviations of 7.9% (n = 7, CPb = 0.02 µg L-1), fast adsorption kinetics (5 min), and strong ability to overcome matrix interference. Validation was also performed by analyzing a certified standard reference material, and the method was successfully applied to real tap water, lake water, human urine, and human blood serum with satisfactory recoveries of 92.6-109%.

Assessment of automated off-line solid-phase extraction LC-MS/MS to monitor EPA priority endocrine disruptors in tap water, surface water, and wastewater.

EPA method 539.1 recently introduced an expanded list of priority endocrine-disrupting compounds (EDCs), some of which were also included in the Unregulated Contaminant Monitoring Rule 3 (UCMR3). Though standardized methods are available for drinking water, analysis of steroid hormones and bisphenol A (BPA) at the ultra-trace level remains challenging. This study set out to evaluate the suitability of automated off-line solid-phase extraction (SPE) liquid chromatography tandem mass spectrometry (UHPLC-MS/MS) for the determination of EPA-priority EDCs in environmental water matrixes (tap water, surface water, and wastewater influents and effluents). The target molecules included 14 steroid hormones (altrenogest, androstenedione, equilenin, equilin, α-estradiol, ß-estradiol, estriol, estrone, ethinylestradiol, levonorgestrel, medroxyprogesterone, norethindrone, progesterone, testosterone) and BPA. Factors that may influence the analytical performance were assessed. This involved, for instance, testing combinations of SPE materials from different brands and protocol variations. Several materials presented absolute extraction efficiencies in acceptable ranges. Initial sample pH, nature of reconstitution medium, and mobile phase salt concentration were among the potential factors affecting analyte signal. Storage conditions (different preservative agents) possibly exerted the strongest influence, in agreement with the literature. Limits of detection were in the range of 0.03-0.5 ng/L in drinking water, 0.1-0.5 ng/L in surface water, and 0.16-1 ng/L in wastewater. Method validation also involved testing linearity, accuracy, and precision in reagent water and matrix-matched extracted calibrants. The method was applied to field-collected water samples in Eastern Canada. Summed EDC concentrations remained low in tap water (

The pesticide residue analysis in commodities with high content of chlorophyll based on the quick, easy, cheap, effective, rugged, and safe method: A review.

In multiresidue analysis, the QuEChERS (Quick, Easy, Cheap, Effective, Rugged, and Safe) is one of the most popular techniques routinely used by researchers during pesticide analysis of food and vegetable samples. Originally, the QuEChERS method was developed for analysis of pesticide residues from fruits and vegetables, but rapidly gained popularity in the extraction of analytes from different matrices. This analytical approach shows several advantages over traditional extraction techniques: it requires lower sample and solvent amounts while shortening the time of sample preparation. However, it presents some limitations for complex matrices such as those containing high amounts of chlorophyll. To overcome the problem of strong matrix effect and influence of interferences, different approaches are applied. Most are concerning modifications of the cleanup step, that is, sorbent type and its amount. Optimization of other parameters, such as sample size, hydration level, extraction solvent, and buffering, also has an impact on overall performance. Combining proper sample preparation with modern highly sensitive and selective detection techniques enables receiving desired limits of quantification. This article presents an overview of strategies employed by researchers for analysis of green, high chlorophyll content commodities and results obtained in their studies.

Green and Cost-Effective Extraction Techniques of Quercetin from Mixture of Nutraceuticals with Yield Analysis via Spectrophotometry and High-Performance Liquid Chromatography Methods.

BACKGROUND: Extraction is the leading critical stage in the analysis of nutraceuticals. Ginkgo biloba (GB) has gained interest because of its therapeutic usages. OBJECTIVES: The aim was to develop four cost-effective extraction techniques for the extraction of quercetin from GB in a sachet containing a mixture of nutraceuticals. These techniques are solid-phase extraction (SPE), liquid-liquid extraction, inverted dispersive liquid-liquid microextraction, and the QuEChERS (quick, easy, cheap, effective, rugged, and safe) method. METHOD: Direct spectrophotometry was used to monitor the recovery of the standard quercetin throughout the optimization steps. The HPLC-UV method of analysis was optimized to quantify the yields from the extracts present in the complicated contents of the sachets. The present study was assessed by analytical Eco-Scale assessment (ESA) and the National Environmental Method Index (NEMI) for greenness in comparison with the literature. RESULTS: SPE showed the best cleanup outcomes. ESA and NEMI showed an adequate greenness of the proposed extraction protocol. CONCLUSIONS: Quercetin (marker for GB) extraction from market nutraceutical sachets is considered an exemplar for analysis in the QC of nutraceuticals. Regarding the greenness results, the proposed method of extraction is better even with adequate greenness as the extraction was a one-step process, in comparison with multistep processes of previously published protocols. Accordingly, it is recommended for use in routine extraction and analysis of such nutraceuticals. HIGHLIGHTS: Four extraction protocols have been developed. For GB ternary-mixture sachets, proper recovery was obtained using C18 SPE. The assessment of greenness of the proposed protocol guaranteed the superiority of the presented method. Safer sorbents and chemicals are favored for use in routine extraction of nutraceuticals.

Approaches to liquid chromatography tandem mass spectrometry assessment of glyphosate residues in wine.

The performance of two different analytical methodologies to investigate the presence of glyphosate (GLY) and aminomethylphosphonic acid (AMPA) residues in wine samples was evaluated. Transformation of compounds in their fluorene-9-methyloxycarbonyl derivatives permitted their separation under reversed-phase liquid chromatography with tandem mass spectrometry (LC-MS/MS) determination. Although the wine matrix severely impaired the efficiency of GLY derivatization, this drawback was solved using a molecularly imprinted sorbent for the previous, selective extraction of GLY and AMPA from wine. Alternatively, the use of a strong anionic exchange, polyvinyl alcohol-based LC column, turned to be the most effective alternative for direct determination of both compounds in diluted wine samples. The chromatographic behavior of this column and the magnitude of matrix effects observed during analysis of diluted wine samples were significantly affected by the composition of the mobile phase. Under final working conditions, this column permitted the separation of AMPA and the fungicide fosetyl (which shows common transitions in tandem MS/MS methods), it improved significantly the sample throughput versus extraction-derivatization-purification method, and it allowed the use of solvent-based calibration standards. Both analytical procedures provided similar limits of quantification (LOQs) for GLY (0.5-1.0 ng mL-1), while the multistep method was 8 times more sensitive to AMPA than the direct procedure. GLY residues stayed above method LOQs in 70% of the processed wines; however, concentrations measured in 95% of positive samples remained 100 times below the maximum residue limit (MRL) set for GLY in vinification grapes.

Rapid quantification of fatty acids in plant oils and biological samples by LC-MS.

Analysis of fatty acids (FA) in food and biological samples such as blood is indispensable in modern life sciences. We developed a rapid, sensitive and comprehensive method for the quantification of 41 saturated and unsaturated fatty acids by means of LC-MS. Optimized chromatographic separation of isobaric analytes was carried out on a C8 reversed phase analytical column (100 × 2.1 mm, 2.6 µm core-shell particle) with a total run time of 15 min with back pressure lower than 300 bar. On an old triple quadrupole instrument (3200, AB Sciex), pseudo selected reaction monitoring mode was used for quantification of the poorly fragmenting FA, yielding limits of detection of 5-100 nM. Sample preparation was carried out by removal of phospholipids and triglycerides by solid-phase extraction (non-esterified fatty acids in oils) or saponification in iso-propanol (fatty acyls). This is not only a rapid strategy for quantification of fatty acyls, but allows the direct combination with the LC-MS-based analysis of fatty acid oxidation products (eicosanoids and other oxylipins) from the same sample. The concentrations of fatty acyls determined by means of LC-MS were consistent with those from GC-FID analysis demonstrating the accuracy of the developed method. Moreover, the method shows high precisions with a low intra-day (≤ 10% for almost all fatty acids in plasma and ≤ 15% in oils) and inter-day as well as inter-operator variability (< 20%). The method was successfully applied on human plasma and edible oils. The possibility to quantify non-esterified fatty acids in samples containing an excess of triacylglycerols and phospholipids is a major strength of the described approach allowing to gain new insights in the composition of biological samples.

Highly-selective complex matrices removal via a modified QuEChERS for determination of triazine herbicide residues and risk assessment in bivalves.

A modified quick, easy, cheap, effective, rugged, and safe (QuEChERs) method for determining triazine herbicide residues in bivalves (Mussels, Scallops, Cockles) was developed. The use of molecularly imprinted polymers (MIPs) as a selective purification material during dispersive-solid phase extraction (d-SPE) increased the removal rate of pigments interference. With 4% acidic acetonitrile as the organic modifier, the modified QuEChERs method achieved good extraction rate of herbicide residues. The satisfactory recoveries (80%-118%) and RSDs (1.0%-11.6%) of herbicide residues were obtained at three spiked levels. The limits of quantification of herbicide residues ranged from 0.10 µg/kg to 1.59 µg/kg. Further, the herbicide residues in bivalves collected in the eastern coasts of China was analyzed. The developed QuEChERs procedure coupled with GC-MS/MS was successfully applied to the herbicide residues detection in bivalves, and due to the extensive use of herbicides and the large consumption of bivalves in globally, the ongoing risk evaluation is needed.

QuEChERS and 96-well plate solid phase extraction for determination of vancomycin and norvancomycin in fish meat by UPLC-MS/MS.

Vancomycin and norvancomycin are glycopeptide antibiotics for gram-positive bacteria infection, but indiscriminately used in aquaculture. In this study, a QuEChERS (quick, easy, cheap, effective, rugged, and safe)/96-well solid-phase extraction (SPE) plate method was used to extract vancomycin and norvancomycin in fish meat samples, and the drugs were further analyzed by ultra-performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS). The parameters, such as the sorbent of cation exchange resin, the proportion of acetonitrile (15%) in extractant, the mobile phase of water (0.1% formic acid)/acetonitrile, were optimized. The method was validated in terms of linearity (0.9990-0.9994), LOD (0.51 µg·kg-1), LOQ (1.73 µg·kg-1), intra-dayprecision (<5.19%), inter-day precision (<6.30%), and recovery (86.7-98.6%). Finally, the method was successfully applied to contaminated and randomly collected samples. The results indicated that the proposed method meet the daily monitoring requirements for vancomycin and norvancomycin.

UltraPrep is a scalable, cost-effective, bead-based method for purifying cell-free DNA.

UltraPrep is an open-source, two-step method for purification of cell-free DNA that entails extraction of total DNA followed by size-selective enrichment of the smaller fragments that are characteristic of DNA originating from fragmentation between nucleosome. The advantages of the two related protocols that are described are that they can easily accommodate a wide range of sample input volumes, they rely on simple, magnetic bead-based technology, the yields of cfDNA are directly comparable to the most popular methods for cfDNA purification, and they dramatically reduce the cost of cfDNA isolation relative to currently available commercial methods. We provide a framework for physical and molecular quality analysis of purified cfDNA and demonstrate that the cfDNA generated by UltraPrep meets or exceeds the quality metrics of the most commonly used procedure. In addition, our method removes high molecular weight genomic DNA (hmwgDNA) that can interfere with downstream assay results, thereby addressing one of the primary concerns for preanalytical collection of blood samples.

A practical method for preparing fluorescent-labeled glycans with a 9-fluorenylmethyl derivative to simplify a fluorimetric HPLC-based analysis.

Analysis of glycans in glycoproteins is often performed by liquid chromatography (LC) separation coupled with fluorescence detection and/or mass spectrometric detection. Enzymatically or chemically released glycans from glycoproteins are usually labeled by reductive amination with a fluorophore reagent. Although labeling techniques based on reductive amination have been well-established as sample preparation methods for fluorometric HPLC-based glycan analysis, they often include time-consuming and tedious purification steps. Here, we reported an alternative fluorescent labeling method based on the synthesis of hydrazone and its reduction using 9-fluorenylmethyl carbazate (Fmoc-hydrazine) as a fluorophore reagent. Using isomaltopentaose and N-glycans from human IgG, we optimized the Fmoc-labeling conditions and purification procedure of Fmoc-labeled N-glycans and applied the optimized method for the analysis of N-glycans released from four glycoproteins (bovine RNase B, human fibrinogen, human α1-acid glycoprotein, and bovine fetuin). The complete workflow for preparation of fluorescent-labeled N-glycans takes a total of 3.5 h and is simple to implement. The method presented here lowers the overall cost of a fluorescently labeled N-glycan and will be practically useful for the screening of disease-related glycans or routine analysis at an early stage of development of biopharmaceuticals.