Project description:A simple, cost effective, and yet sensitive sample preparation technique was investigated for determining Polycyclic Aromatic Hydrocarbons (PAHs) in solid samples. The method comprises ultrasonic extraction, Stir Bar Sorptive Extraction (SBSE), and thermal desorption-gas chromatography-mass spectrometry to increase analytical capacity in laboratories. This method required no clean-up, satisfied PAHs recovery, and significantly advances cost performance over conventional extraction methods, such as Soxhlet and Microwave Assisted Extraction (MAE). This study evaluated three operational parameters for ultrasonic extraction: solvent composition, extraction time, and sample load. A standard material, SRM 1649 a (urban dust), was used as the solid sample matrix, and 12 priority PAHs on the US Environmental Protection Agency (US EPA) list were analyzed. Combination of non-polar and polar solvents ameliorated extraction efficiency. Acetone/hexane mixtures of 2:3 and 1:1 (v/v) gave the most satisfactory results: recoveries ranged from 63.3% to 122%. Single composition solvents (methanol, hexane, and dichloromethane) showed fewer recoveries. Comparing 20 min with 60 min sonication, longer sonication diminished extraction efficiencies in general. Furthermore, sample load became a critical factor in certain solvent systems, particularly MeOH. MAE was also compared to the ultrasonic extraction, and results determined that the 20-min ultrasonic extraction using acetone/hexane (2:3, v/v) was as potent as MAE. The SBSE method using 20 mL of 30% alcohol-fortified solution rendered a limit of detection ranging from 1.7 to 32 ng L(-1) and a limit of quantitation ranging from 5.8 to 110 ng L(-1) for the 16 US EPA PAHs.
Project description:A new analytical approach to the simultaneous identification and quantification of vitamins A and E in three representative matrices (Parmesan, spinach, and almonds) was developed. The analyses were based on high-performance liquid chromatography with UV-VIS/DAD detection. The procedure was optimized by a significant reduction in the weight of the tested products and quantities of reagents added during the saponification and extraction stages. A full method validation study was performed for retinol at two concentration levels (LOQ and 200 × LOQ), which showed satisfactory results, with recoveries ranging from 98.8 to 110.1%, and an average CV of 8.9%. Linearity was tested in the range of 1-500 µg/mL and showed the coefficient of determination R2 = 0.999. The satisfactory recovery and precision parameters were achieved for α-tocopherol (LOQ and 500 × LOQ) in the range of 70.6-143.2%, with a mean CV equal to 6.5%. The observed linearity for this analyte in the concentration range of 1.06-532.0 µg/mL was R2 = 0.999. The average extended uncertainties were estimated, using a top-down approach of 15.9% and 17.6% for vitamin E and A, respectively. Finally, the method was successfully applied to determine vitamins in 15 commercial samples.
Project description:Placental elemental composition can serve as an indicator for neonatal health. Medical studies aiming at revealing such cause-and-effect relationships or studies monitoring potential environmental influences consist of large sample series to ensure statistically sufficient data. Several analytical techniques have been used to study trace metals in human placenta. However, most techniques require provision of clear liquid sample solutions and therefore time- and reagent-consuming total digestion of biological tissue is necessary. In total reflection X-ray fluorescence spectrometry (TXRF)-a straightforward multielement analytical technique-in contrast suspensions of minute sample amounts can be analyzed directly. Therefore, herein we report on a valid method to prepare homogenous sample suspensions for sustainable and fast TXRF analysis of large sample series. The optimized method requires only 10 mg of powdered placental tissue and 1 mL nitric acid. Suspensions are readily prepared within 30 min and the found mass fractions of major, minor, and trace elements are in good agreement in comparison to analysis of digests. In addition, possible effects on fixation time and the exact sampling location, i.e., maternal vs. fetal side of the placenta, were studied applying this method. Thereby, significant differences for fetal placenta tissue compared to maternal or intermediate tissue were observed revealing accumulation of trace elements in the fetal side of the placenta. Furthermore, considerable depletion of up to 60% mass fraction with longer fixation duration occurred in particular in fetal placenta tissue. These findings help to understand the large ranges of mass fraction of elements in placenta reported in the literature and at the same time indicate the necessity for more systematic investigation of non-homogenous elements distributed in placenta taking sampling and stabilization methods into account.
Project description:A successful mass spectrometry-based phosphoproteomics analysis relies on effective sample preparation strategies. Suspension trapping (S-Trap) is a novel, rapid, and universal method of sample preparation that is increasingly applied in bottom-up proteomics studies. However, the performance of the S-Trap protocol for phosphoproteomics studies is unclear. In the existing S-Trap protocol, the addition of phosphoric acid (PA) and methanol buffer creates a fine protein suspension to capture proteins on a filter and is a critical step for subsequent protein digestion. Herein, we demonstrate that this addition of PA is detrimental to downstream phosphopeptide enrichment, rendering the standard S-Trap protocol suboptimal for phosphoproteomics. In this study, the performance of the S-Trap digestion for proteomics and phosphoproteomics is systematically evaluated in large-scale and small-scale samples. The results of this comparative analysis show that an optimized S-Trap approach, where trifluoroacetic acid is substituted for PA, is a simple and effective method to prepare samples for phosphoproteomics. Our optimized S-Trap protocol is applied to extracellular vesicles to demonstrate superior sample preparation workflow for low-abundance, membrane-rich samples.
Project description:In the present work, we developed a simple and rapid sample preparation method for the determination of neonicotinoid pesticides in honey based on the matrix-induced sugaring-out. Since there is a high concentration of sugars in the honey matrix, the honey samples were mixed directly with acetonitrile (ACN)-water mixture to trigger the phase separation. Analytes were extracted into the upper ACN phase without additional phase separation agents and injected into the HPLC system for the analysis. Parameters of this matrix-induced sugaring-out method were systematically investigated. The optimal protocol involves 2 g honey mixed with 4 mL ACN-water mixture (v/v, 60:40). In addition, this simple sample preparation method was compared with two other ACN-water-based homogenous liquid-liquid extraction methods, including salting-out assisted liquid-liquid extraction and subzero-temperature assisted liquid-liquid extraction. The present method was fully validated, the obtained limits of detection (LODs) and limits of quantification (LOQs) were from 21 to 27 and 70 to 90 μg/kg, respectively. Average recoveries at three spiked levels were in the range of 91.49% to 97.73%. Precision expressed as relative standard deviations (RSDs) in the inter-day and intra-day analysis were all lower than 5%. Finally, the developed method was applied for the analysis of eight honey samples, results showed that none of the target neonicotinoid residues were detected.
Project description:Salmonella is recognized as one of the most important foodborne bacteria and has wide health and socioeconomic impacts worldwide. Fresh pork meat is one of the main sources of Salmonella, and efficient and fast methods for detection are therefore necessary. Current methods for Salmonella detection in fresh meat usually include >16 h of culture enrichment, in a few cases <12 h, thus requiring at least two working shifts. Here, we report a rapid (<5 h) and high-throughput method for screening of Salmonella in samples from fresh pork meat, consisting of a 3-h enrichment in standard buffered peptone water and a real-time PCR-compatible sample preparation method based on filtration, centrifugation, and enzymatic digestion, followed by fast-cycling real-time PCR detection. The method was validated in an unpaired comparative study against the Nordic Committee on Food Analysis (NMKL) reference culture method 187. Pork meat samples (n = 140) were either artificially contaminated with Salmonella at 0, 1 to 10, or 10 to 100 CFU/25 g of meat or naturally contaminated. Cohen's kappa for the degree of agreement between the rapid method and the reference was 0.64, and the relative accuracy, sensitivity, and specificity for the rapid method were 81.4, 95.1, and 97.9%, respectively. The 50% limit of detections (LOD50s) were 8.8 CFU/25 g for the rapid method and 7.7 CFU/25 g for the reference method. Implementation of this method will enable faster release of Salmonella low-risk meat, providing savings for meat producers, and it will help contribute to improved food safety.IMPORTANCE While the cost of analysis and hands-on time of the presented rapid method were comparable to those of reference culture methods, the fast product release by this method can provide the meat industry with a competitive advantage. Not only will the abattoirs save costs for work hours and cold storage, but consumers and retailers will also benefit from fresher meat with a longer shelf life. Furthermore, the presented sample preparation might be adjusted for application in the detection of other pathogenic bacteria in different sample types.
Project description:Amyloids share a common architecture but play disparate biological roles in processes ranging from bacterial defense mechanisms to protein misfolding diseases. Their structures are highly polymorphic, which makes them difficult to study by X-ray diffraction or NMR spectroscopy. Our understanding of amyloid structures is due in large part to recent advances in the field of cryo-EM, which allows for determining the polymorphs separately. In this review, we highlight the main stepping stones leading to the substantial number of high-resolution amyloid fibril structures known today as well as recent developments regarding automation and software in cryo-EM. We discuss that sample preparation should move closer to physiological conditions to understand how amyloid aggregation and disease are linked. We further highlight new approaches to address heterogeneity and polymorphism of amyloid fibrils in EM image processing and give an outlook to the upcoming challenges in researching the structural biology of amyloids.
Project description:Cost-effectiveness models are commonly utilized to determine the combined clinical and economic impact of one treatment compared to another. However, most methods for sample size determination of cost-effectiveness studies assume fully observed costs and effectiveness outcomes, which presents challenges for survival-based studies in which censoring exists. We propose a Bayesian method for the design and analysis of cost-effectiveness data in which costs and effectiveness may be censored, and the sample size is approximated for both power and assurance. We explore two parametric models and demonstrate the flexibility of the approach to accommodate a variety of modifications to study assumptions.
Project description:Asphaltenes constitute the heaviest, most polar and aromatic fraction of petroleum crucial to the formation of highly-stable water-in-crude oil emulsions. The latter occur during crude oil production as well as spills and cause difficulties to efficient remediation practice. It is thought that in nanoaggregate form, asphaltenes create elastic layers around water droplets enhancing stability of the emulsion matrix. Ultrasonic characterisation is a high-resolution non-invasive tool in colloidal analysis shown to successfully identify asphaltene nanoaggregation in toluene. The high sensitivity of acoustic velocity to molecular rearrangements and ease in implementation renders it an attractive method to study asphaltene phase properties. Currently, aggregation is thought to correspond to an intersection of two concentration-ultrasonic velocity regressions. Our measurements indicate a variation in the proximity of nanoaggregation which is not accounted for by present models. We attribute this uncertainty to physico-chemical heterogeneity of the asphaltene fraction driven by variation in molecular size and propose a critical nanoaggregation region. We treated asphaltenes from North and South American crude oils with ruthenium ion catalysed oxidation to characterize their n-alkyl appendages attached to aromatic cores. Principal component analysis was performed to investigate the coupling between asphaltene structures and velocity measurements and their impact on aggregation.