Extraction Reagents Research Articles

Rapid and highly sensitive detection of trace chromium and copper in tea infusion using laser-induced breakdown spectroscopy combined with electrospinning technology

Tea infusion, as one of the most popular beverages, is favored by people around the world. However, tea plants accumulate heavy metals from the soil during growth, which are gradually transferred to the leaves and subsequently decomposed into the tea infusion after soaking in hot water. Long-term consumption of tea infusion containing heavy metals may lead to severe health issues, such as liver dysfunction and cancer. Therefore, rapid, precise, and highly sensitive detection of heavy metals in tea infusion becomes crucial for human health. Traditional methods, such as atomic absorption spectroscopy (AAS), suffer from long detection cycles, high costs, and the risk of secondary pollution from chemical extraction reagents. Thus, this paper presents a method, using laser-induced breakdown spectroscopy combined with electrospinning technology (LIBS-ES), to detect trace amounts of Cr and Cu in tea infusion. The electrospun nanofiber membranes (ENM) were modified with gold nanoparticles (AuNPs) and silver nanoparticles (AgNPs), achieving ultra-sensitive detection of Cr and Cu. The experiment results show that the limits of detection for Cr and Cu are 5 μg/L and 10 μg/L, respectively. Moreover, this method was successfully applied to the detection of Cr and Cu in seven different types of tea infusion, achieving recovery rates of 99 %-106 % and 99 %-108 %, respectively. The method demonstrated good performance even under the interference of other ions at various concentrations, providing a reference for the rapid and highly sensitive detection of heavy metals in tea infusion.

A multicommuted flow system with a solid–liquid extraction chamber to extract and quantify total phenolic compounds from solid samples

Multicommutation significantly enhances the versatility of flow systems by integrating solenoid micro-pumps or valves. In this novel approach, the flow system was fitted with four solenoid micro-pumps to enhance mixing conditions, minimize reagent use and waste production, and quantify total phenolic compounds (TPC) via the Folin-Ciocalteu method. The extraction module was constructed with a 1000 µL pipette tip, a frit, and cotton used as a filter. The fluidized bed column was continuously agitated with a vibratory shaker, the extraction solution (ethanol 5 % (v/v)), and the solid samples (50 mg). The extraction of TPC was started by pumping the extraction solution through P1 (150 pulses). The analytical cycle was established by delivering the extraction solution and reagents through P1 (4 pulses), P3 (Folin-Ciocalteu 20 % (v/v); 1 pulse), and P4 (sodium carbonate 2 % (w/v); 1 pulse). These aliquots were rapidly mixed at the interfaces, initiating the first sampling cycle. This process was repeated seven times to create the sample zone and was directed towards detection at 740 nm by activating P2. Under optimized experimental conditions, a linear response was observed from 1 to 20 mg L-1 gallic acid, as described by the equation Absorbance = 0.023 + 0.028C (mg L-1), r = 0.997. At a 99.7 % confidence level, the detection limit, coefficient of variation (n = 11; 10 mg L-1), and sampling rate were determined to be 188 µg L-1, 0.1 %, and 100 determinations per hour, respectively. The consumption of reagents for each determination was estimated at 0.95 mg of sodium carbonate and 9.5 µL of Folin-Ciocalteu, resulting in 3.4 mL of waste generated. Phenolic compounds were quantified between 694 and 1760 mg in 100 g−1 food flour dry samples. Results obtained by the proposed process agreed with those obtained with batch extractions (95 % confidence level). Typical characteristics of systems that use solenoid micro-pumps are portability, cost-effectiveness, low energy consumption, improved mixing, and reproducibility in handling micro-volume solutions. The proposed procedure demonstrates its versatility and effectiveness by offering a practical alternative for extracting and quantifying total phenolic compounds in various solid matrices. This approach proves to be a superior option compared to existing methodologies.

B-156 Extraction strategy for therapeutic drug monitoring of sirolimus and tacrolimus using volumetric absorptive microsampling

Abstract Background Following organ transplantation, patients are placed on immunosuppressant drugs (ISDs) to minimize the risk of rejection. Sirolimus and tacrolimus are two of the most widely used ISDs, however, due to their narrow therapeutic indices, patients require frequent therapeutic drug monitoring (TDM). Recurrent venipuncture can become uncomfortable and disruptive, especially in the pediatric population. Therefore, we describe an extraction method using dried whole blood from volumetric absorptive microsampling (VAMS) devices for the detection of sirolimus and tacrolimus by turbulent flow liquid chromatography tandem mass spectrometry (TFC-MS/MS). The use of VAMS in the clinical laboratory will provide ISD patients with a convenient alternative to traditional blood draws. Methods MassTrak immunosuppressant calibrators based on a whole blood matrix (Waters) were applied to 30 μL VAMS Mitra devices (Neoteryx) and dried overnight. To extract the whole blood off of VAMS tip, we tested various methanol:water solvent ratios, of which 30 μL was added to the device. Tips were extracted using ultrasonication (30 min), vortexing (5 min), and centrifugation. Once extracted from the tip, 25 mM zinc sulfate was added to whole blood extract at a 1:1 (v/v) ratio. An acetonitrile and methanol extraction reagent was then added to the whole blood/zinc sulfate solution at a 2:1 (v/v) ratio. After brief vortexing, samples were centrifuged at 16,060 × g (10 min) and tested using an established TFC-MS/MS method which deploys a TLX-2 system for sample preparation coupled to a Q-Trap tandem mass spectrometer (AB Sciex 5500). Results A 80:20 (v/v) water:methanol extraction solvent yielded the greatest extraction efficacy for both sirolimus and tacrolimus from the VAMS tips compared to other tested ratios. We established a 5-point calibration curve to correlate the measured values of ISDs with known MassTrak calibration standard concentrations. The resulting average calibration curve from 3 independent experiments was highly linear for both sirolimus (R2= 0.992; slope= 1.1; intercept= 0.94) and tacrolimus (R2= 0.996; slope= 1.1; intercept = 0.8819). Our VAMS whole blood extraction strategy was correlated to the established neat whole blood protocol, which utilized all of the sample preparation steps outlined above except the initial extraction from the VAMS tip. The average extraction efficacy was 89.1% for sirolimus with an average coefficient of variation (CV%) of 12.1% across 3 independent experiments. For tacrolimus, we demonstrated an average extraction efficacy of 100.1% with an average CV% of 8.4%, again across 3 independent experiments. Conclusions We demonstrate the feasibility of using a VAMS approach to TDM of sirolimus and tacrolimus. Our results demonstrate favorable recoveries for both of the tested drugs, evidenced by high recoveries of the external calibration standards. Additionally, our method exhibited high extraction efficacy and low variability. Further validation studies, including the utilization of clinical samples is necessary to confirm our preliminary findings.

Highly selective extraction of aluminum from chloride and sulfate solutions using a mixture of alkoxycarboxylic acid and tributyl phosphate

Four different solvent extraction systems are compared for aluminum (Al) separation in the presence of alkali metals, alkaline-earth metals, and transition metals. The octoxyacetic acid (OOA acid) is utilized as an Al extraction reagent for the first time, exhibiting high selectivity for Al. The impacts of the concentration of extractants, the equilibrium pH of the aqueous phase, and the concentration of phase modifier were studied systemically. The results indicate that the Al extraction efficiency using OOA/TBP (tributyl phosphate) system could reach 98.9 ± 0.63 % via a single-step solvent extraction process while maintaining negligible extraction for the other metal ions at the pH value of ∼ 4.2. Meanwhile, the separation factors (β) of Al over magnesium, calcium, manganese, cobalt and nickel reach the maximum values (βAl/Mg = 285157, βAl/Ca = 18275, βAl/Mn = 19271, βAl/Co = 10814, βAl/Ni = 5969). Slope analysis and infrared spectroscopic characterization confirm that the extraction of Al by the OOA/TBP system follows the cation exchange mechanism. The OOA/TBP solvent extraction system is promising for the efficient separation of Al, with potential applications in a wide range of practical scenarios.

Rapid discovery of natural antioxidants in Hypericum japonicum: Dual roles of the liquid phase mobile phase as extraction and separation solvent

Hypericum japonicum is a traditional folk medicine with various bioactivities such as hepatoprotective, antioxidant, and anti-tumorous. The antioxidant effect of H. japonicum is one of the most prominent effects due to its responsibility for many of its activities. To clarify active natural substance, the antioxidant properties of H. japonicum were preliminarily assessed by ferric reducing–antioxidant power (FRAP), 2,2-azino-bis-3-ethylbenzothiazoline-6-sulphonic acid (ABTS) and Oxygen radical absorbance capacity (ORAC), as well as superoxide dismutase (SOD). Then, a straightforward and effective method named online liquid extraction-high performance liquid chromatography combined with ABTS antioxidant assay and mass spectrometry (OLE-HPLC-ABTS/Q-TOF-MS) was developed to swiftly and directly discover the antioxidants in H. japonicum. Using mobile phase as extraction and separation reagent, coupled with online activity analysis and compounds identification by high-resolution MS, the online system enables rapid screening of natural antioxidant bioactives from complex mixture. By using it, a total of 9 compounds including flavonoids and phenolic acids characterized by retention time, precise mass, and fragmentation ions in MS/MS spectra showed antioxidant action. Finally, the antioxidant and SOD activity of main found active compounds were validated by in vitro experiment assay and molecular docking. In summary, these results suggested that H. japonicum could be considered as a potential source of natural antioxidants, and the online integrated system might become a promising candidate for the natural antioxidants discovery in the future.

A low-cost and versatile paramagnetic bead DNA extraction method for Mycobacterium ulcerans environmental surveillance.

Buruli ulcer (BU) is a neglected tropical skin disease, with an incidence that has dramatically increased in temperate southeastern Australia over the last decade. In southeastern Australia, BU is a zoonosis with native possums the major wildlife reservoir of the causative pathogen, Mycobacterium ulcerans. Infected possums shed M. ulcerans in their excreta, and excreta surveys using PCR to screen for the presence of pathogen DNA are a powerful means to predict future areas of Buruli ulcer risk for humans. However, excreta surveys across large geographic areas require testing of many thousands of samples. The cost of commercial DNA extraction reagents used for preparing samples for PCR testing can thus become prohibitive to effective surveillance. Here, we describe a simple, low-cost method for extracting DNA from possum excreta using paramagnetic beads. The method is versatile and adaptable to a variety of other sample types including swabs collected from possum tissues and pure cultures of mycobacteria.

Synthesis of New Dithiocarbamate Derivative Reagent for Micro Extraction and Determination of Iron(II) Ion in Aqueous Solution

Synthesis of New Dithiocarbamate Derivative Reagent for Micro Extraction and Determination of Iron(II) Ion in Aqueous Solution

Sample-in-answer-out centrifugal microfluidic chip reaction biosensor powered by Thermus thermophilus Argonaute (TtAgo) for rapid, highly sensitive and multiplexed molecular diagnostics of foodborne bacterial pathogens

Foodborne pathogens endanger public health and rapid, sensitive and accurate detection of them is vital. Argonaute stands in the frontline as the next generation nucleic acid detection tool, bearing a few comparable advantages. Centrifugal microfluidic chips (CMCs) use centrifugal force to achieve liquid flowing, mixing and reaction, eliminating complicated designs of valves and pumps. In this study, for the first time, we devised a Thermus thermophilusArgonaute (TtAgo)-powered centrifugal microfluidic chip reaction biosensor for Sample-in-Answer-out detection of Pathogen S. aureus (termed as ASAP) with ultrahigh rapidity and sensitivity in a one-pot fashion. The samples subjected to testing were mixed with a home-made nucleic acid fast extraction reagent and then the mixture was injected into the sample cell of CMC, which was centrifuged down into the reaction cell. The reaction cell was preloaded with both LAMP (Loop-mediated isothermal amplification) and TtAgo systems. As such, the species-specific nuc genes were amplified by LAMP, while the TtAgo performed site-specific cleavage to output fluorescent (FL) signals. The sample-to-answer time was 17 min, and the limit of detection (LOD) reached 1 CFU/mL. ASAP was able to perform simultaneous multiplexed detection and up to 16 samples can be detected at the same time. ASAP reduced reagent consumption and minimized the influence of carry-over and cross-contamination. ASAP was capable of detecting S. aureus in foods, but also detecting physiological samples from infect, holding great promise for practical applications. Overall, our work has enriched the Argonaute-powered biosensing and CMC biosensor technology by providing a conceptually novel bacterial detection platform.

Extraction and Hemagglutination Activity Study of Lectins from Hygrophorus lucorum Kalchbr

Edible fungus lectin hasanti-tumor, anti-virus, immune regulation and other functions. In this experiment, the wild Hygrophorus lucorum Kalchbr was used as the material, and the lectin was extracted by leaching method. The lectin was treated with physical and chemical factors, and the hemagglutination method was used to detect the change rule of lectin agglutination activity. The extraction process of lectin was optimized by single factor experiment and orthogonal experiment. The study of agglutination activity showed that: glycerol, lactose and maltose had significant inhibitory effects; when treated at 80℃, the agglutination activity was lost; pH<5.0 or pH>9.0, the agglutination activity was greatly inhibited; AI3+, Fe3+ had obvious promoting effects. Lectin extraction single factor test results: PBS (0.50mol/L, pH6.5) was used as the extraction reagent, the ratio of solid to liquid(g/mL) was1:10, and the fruiting bodies were extracted for 5 hours at 30℃. 40% saturation ammonium sulfate salting-out treatment, the salting-out product has the highest agglutination activity. The optimal process combination obtained by the or thogonal test is: using PBS (0.50mol/L, pH 6.5) as the extractant, according to the solid-liquid ratio (g/mL) 1:10, 40℃, leaching for 4h, the quantitative agglutination activity of lectin was 0.9609.

A modified dithionite reduction method for the quantification of iron-bound organic carbon in marine sediments

Iron-bound organic carbon (Fe-OC) plays a significant role in the global marine carbon cycle, facilitating the long-term accumulation and storage of organic carbon. However, the conventional method for quantifying Fe-OC, known as the citrate-bicarbonate-dithionite (CBD) method, has limitations in terms of its efficiency in extracting both Fe-OC and iron (Fe). Another CBD method, commonly used for Fe speciation analysis in paleoenvironmental reconstructions, has shown efficient and selective extraction of Fe oxides; however, its effectiveness in extracting Fe-OC remains uncertain. To evaluate the efficiency of the two CBD methods, synthetic samples spiked with organic acid/environmental organic matter‑iron (hydr)oxide coprecipitates and a marine sediment sample were subjected to extraction. The CBD approach for Fe speciation analysis at pH 4.8 on spiked samples demonstrated higher efficiency compared to the conventional CBD method, but not for samples of marine sediment. This result suggests that the synthetic Fe-OC in the spiked samples did not accurately represent the realistic state of Fe-OC in marine sediments. We found that the pH of the leaching solution during the CBD extraction significantly influenced the efficiency of the Fe-OC and Fe extraction. Our tests at pH 4.8, 5.5, 6.0, 6.5, and 7.0 revealed that lower pH enhanced the extraction of Fe, but hindered the release of Fe-OC. Our study revealed that the new CBD (pH 6.5) method resulted in approximately 30% higher yields of Fe-OC and Fe compared to the conventional CBD method, despite of significant variability in the Fe-OC data. The modified CBD (pH 6.5) method uses lower concentration of extraction reagent and reaction temperature, both of which is considered beneficial. Therefore, we suggest that the new CBD method (pH 6.5) is at least of similar efficiency as the conventional method, and can be employed alongside the established method to facilitate the evaluation of the role of Fe-OC in the marine carbon cycle.

A SAW-Based Programmable Controlled RNA Detecting Device: Rapid In Situ Cytolysis-RNA Capture-RNA Release-PCR in One Mini Chamber.

Viral RNA detection is crucial in preventing and treating early infectious diseases. Traditional methods of RNA detection require a large amount of equipment and technical personnel. In this study, proposed a programmable controlled surface acoustic wave (SAW)-based RNA detecting device has been proposed. The proposed device can perform the entire viral RNA detection process, including cell lysis by cell-microparticle collision through SAW-induced liquid whirling, RNA capture by SAW-suspended magnetic beads, RNA elution through SAW-induced high streaming force, and PCR thermal cycling through SAW-generated heat. The device has completed all RNA detection steps in one mini chamber, requiring only 489 µl reagents for RNA extraction, much smaller than the amount used in manual RNA extraction (2065 µl). The experimental results have shown that PCR results from the device are comparable to those achieved via commercial qPCR instrumental detection. This work has demonstrated the potential of SAW-based lab-on-a-chip devices for point-of-care testing and provided a novel approach for rapidly detecting infectiousdiseases.

BSEO-Semiautomatic Method for Determination of Oil Recovery with Nanofluids in Microfluidic Devices.

Microfluidic models have become essential instruments for studying enhanced oil recovery techniques through fluid and chemical injection into micromodels to observe interactions with pore structures and resident fluids. The widespread use of cost-effective lab-on-a-chip devices, known for efficient data extraction and minimal reagent usage, has driven demand for efficient data management methods crucial for high-performance data and image analyses. This article introduces a semiautomatic method for calculating oil recovery in polymeric nanofluid flooding experiments based on the background subtraction (BSEO). It employs the background subtraction technique, generating a foreground binary mask to detect injected fluids represented as pixel areas. The pixel difference is then compared to a threshold value to determine whether the given pixel is foreground or background. Moreover, the proposed method compares its performance with two other representative methods: the ground truth (manual segmentation) and Fiji-ImageJ software. The experiments yielded promising results. Low values of mean-squared error (MSE), mean absolute error (MAE), and root-mean-squared error (RMSE) indicate minimal prediction errors, while a substantial coefficient of determination (R2) of 98% highlights the strong correlation between the method's predictions and the observed outcomes. In conclusion, the presented method emphasizes the viability of BSEO as a robust alternative, offering the advantages of reduced computational resource usage and faster processing times.

MicroRNA extraction using commercial RNA extraction reagent and miRNA extraction effect enhancement solution

MicroRNA extraction using commercial RNA extraction reagent and miRNA extraction effect enhancement solution

Solvent Extraction of Silver(I) using N,N-Diethylacetamido Substituted p-t-Octylcalix[4]arene in Nitrate Media

Solvent extraction of silver ions using tetrakis(N,N-diethylaminocarbonylmethoxy)-p-t-octylcalix[4]arene (EATOC) in chloroform from nitrate medium is presented. The influences on the extraction conditions affecting the behaviour of extraction depending upon the factors such as concentrations of acids, extractants and silver ions as well as extraction time were investigated. Various stripping reagents for back extraction to recover the loaded silver ions by forward extraction process were applied. The extraction mechanism of silver ions using EATOC was established on the 1H NMR chemical shift of the extractant as well as the slope assessments of extraction conditions both before and after loading the silver ions.

Precise Determination of Selenium Forms and Contents in Selenium-Enriched Rapeseed Seedlings and Flowering Stalks by HPLC-ICP-MS.

Rapeseed (Brassica napus L.) has the ability of selenium (Se) enrichment. Identification of selenides in Se-rich rapeseed products will promote the development and utilization of high value. By optimizing the Se species extraction process (protease type, extraction reagent, enzyme sample ratio, extraction time, etc.) and chromatographic column, an efficient, stable, and accurate method was established for the identification of Se species and content in rapeseed seedlings and flowering stalks, which were cultured by inorganic Se hydroponics. Five Se compounds, including selenocystine (SeCys2), methylselenocysteine (MeSeCys), selenomethionine (SeMet), selenite (SeIV), and selenate (SeVI) were qualitatively and quantitatively identified. Organoselenium was absolutely dominant in both seedlings and flowering stalks among the detected rapeseed varieties, with 64.18-90.20% and 94.38-98.47%, respectively. Further, MeSeCys, a highly active selenide, predominated in rapeseed flowering stalks with a proportion of 56.36-72.93% and a content of 1707.3-5030.3 μg/kg. This study provides a new source of MeSeCys supplementation for human Se fortification.

Accelerated Development of a COVID-19 Lateral Flow Test in an Academic Setting: Lessons Learned.

The COVID-19 pandemic further demonstrated the need for usable, reliable, and cost-effective point-of-care diagnostics that can be broadly deployed, ideally for self-testing at home. Antigen tests using more-detectable reporter labels (usually at the cost of reader complexity) achieve better diagnostic sensitivity, supporting the value of higher-analytical-sensitivity reporter technologies in lateral flow.We developed a new approach to simple, inexpensive lateral flow assays (LFAs) of great sensitivity, based on the glow stick peroxyoxalate chemistry widely used in emergency settings and in children's toys. At the peak of the COVID-19 pandemic, we had the opportunity to participate in the pandemic-driven NIH Rapid Acceleration of Diagnostics (RADx) initiative aiming to develop a deployable lateral flow diagnostic for SARS-CoV-2 nucleoprotein based on our novel glow stick-inspired light-emitting reporter technology. During this project, we screened more than 250 antibody pairs for analytical sensitivity and specificity directly in LFA format, using recombinant nucleoprotein and then gamma-irradiated virions spiked into negative nasal swab extracts. Membranes and other LFA materials and swabs and extraction reagent components also were screened and selected. Optimization of conjugate preparation and spraying as well as pretreatment/conditioning of the sample pad led to the final optimized LFA strip. Technology development also included optimization of excitation liquid enclosed in disposable droppers, design of a custom cartridge and smartphone-based reader, and app development, even a prototype reader usable with any mobile phone. Excellent preclinical performance was first demonstrated with contrived samples and then with leftover clinical samples. Moving beyond traditional academic focus areas, we were able to establish a quality management system (QMS), produce large numbers of customized LFA cassettes by contract injection molding, build in-house facilities to assemble and store thousands of complete tests for verification and validation and usability studies, and source kitting/packaging services and quality standard reagents and build partnerships for clinical translation, regulatory guidance, scale up, and market deployment. We were not able to bring this early stage technology to the point of commercialization within the limited time and resources available, but we did achieve strong proof-of-concept and advance translational aspects of the platform including initial high-performance LFAs, reading by the iPhone app using only a $2 plastic dark box with no lens, and convenient, usable excitation liquid packaging in droppers manufacturable in very large numbers.In this Account, we aim to provide a concise overview of our 18-month sprint toward the practical development of a deployable antigen lateral flow assay under pandemic conditions and the challenges and successes experienced by our team. We highlight what it takes to coach a technically savvy but commercially inexperienced academic team through the accelerated translation of an early stage technology into a useful product. Finally, we provide a guided tutorial and workflow to empower others interested in the rapid development of translatable LFAs.

Response of Macroscopic Coal Oxidation and Pyrolysis Parameters to Different Extractants

ABSTRACT The active structure of coal molecules is one of the key factors affecting the characteristics of coal spontaneous combustion (CSC). By actively stripping or reducing the key active structure of coal molecules, and then studying the influence of different structures on the oxidation characteristics of coal, it is helpful to accurately and efficiently inhibit CSC. Herein, using coal samples from Caojiatan Mine in Shaanxi Province, China, the CSC characteristics of 10 coal groups treated with different solvents and the response of the macroscopic coal oxidation and pyrolysis parameters to each extractant were investigated. The extractant type considerably affected CSC, and the oxidization of coal was most responsive to biomass extraction agents, which had the best weakening effect on CSC. The order of CSC characteristics of extracted coal from strong to weak is AE> YM> CYC> AOT> CYH> EG> MT> TP> THF> CYC + AOT + AE> EG + TP. In general, all extraction reagents in each group have a certain weakening effect on the CSC activity of coal samples. The extraction of aromatic hydrocarbon structures can effectively delay the oxidation of coal. The study provides new insights for the development of CSC mitigation technology and have important practical significance for active prevention and control of CSC risks.

Analytical Methods for Extraction of Microplastics from Compost in Sri Lanka

Microplastics (MP) are the plastics with 1 μm-5 mm dimensions. Meanwhile, compost has been identified as a major source of MP in the soil, although compost is a great plant nutrient source, and it provides tremendous benefits for crops. This is primarily ascribed to most of the compost being produced through Municipal Solid Waste Management projects. However, due to the detrimental impacts on soil’s biological, physical, and chemical health, together with the high accumulation probability of MP caused by repeated application of compost, regulations should be implemented on the limitation of MP in compost. Even though there are a few different methods available in the literature for MP extraction and quantification in organic matter (OM)-rich matrices, like compost, sludge, and sediments, a properly validated, routine analytical procedure is missing in Sri Lanka Standards (SLS). Therefore, in this project, the effectiveness of four different MP extraction methods in the literature were compared and validated for compost, to implement an MP analysis method suitable for compost specifications in the SLS. The methodologies included MP extraction by density separation using NaCl or ZnCl2 solutions followed by digestion of OM using 30% KOH: NaOCl solution or Fenton’s reagent. The common MP analysis method for the four protocols was staining with the hydrophobic, fluorescent stain Nile Red, and visual observation. Digestion efficiency of OM and spike recovery of Polystyrene (PS), Polyvinyl Chloride (PVC), Polyethylene terephthalate (PET), High-Density Polyethylene (HDPE), and Polypropylene (PP) microplastics in 1-2 mm dimension by the four MP extraction methods were compared for the validation. T-tests (confidence level=95%) were carried out to determine the significant difference between the OM digestion efficiency in each of the four methods. Each of the combinations compared was significantly different from the other except for ZnCl2/Fenton and NaCl/30% KOH:NaOCl comparison. Meanwhile, the OM removal by ZnCl2/Fenton and NaCl/30% KOH:NaOCl excluding the density separation step also was not significantly different. The spike recovery analysis showed ZnCl2/Fenton combination as the best out of the tested combinations with above 95% recovery percentages for PS, PVC, PP and 100 for HDPE and PET by particle count. However, precipitate formations in the ZnCl2/Fenton combination might cause false negative or positive results for spike recovery when applied to the real samples with smaller MP. The extraction reagents, NaCl was not an ideal solution and ZnCl2 was a good solution for the MP types used. Moreover, in this project, double digestion of OM with NaCl/30% KOH:NaOCl before and after the density separation with ZnCl2 is suggested as a novel and more effective OM digestion protocol before Nile Red staining, and further validations are suggested prior to its final implementation.
 
 Keywords: Microplastics, Compost, Extraction, Organic matter

Effect of Extraction Procedure Substitution on Automated Tacrolimus, Sirolimus, and Cyclosporine Assays.

An erroneously high tacrolimus level was reported to a clinician. A root cause analysis investigation failed to determine the cause of the error. It was suspected that the incorrect preanalytical extraction reagent and procedure was used during testing; however, how this would affect the assayed drug concentration was unclear. Here we investigated the effect of the substitution of sirolimus, tacrolimus, and cyclosporine extraction reagents on assayed drug concentration. Tacrolimus, sirolimus, and cyclosporine concentration were measured on the Abbott Architect i2000 analyzer. Each assay requires a preanalytical extraction step, with a distinct reagent. We investigated the effect of the substitution of the extraction reagents and procedure between the 3 assays on the measured drug concentration. Two experiments were performed, one on samples of known drug concentration and one on samples with no drug present. Substituting cyclosporine and sirolimus extraction procedures increased assayed tacrolimus concentrations from 5.6 to 8.47 (+51.25%) and 8.13 (+45.18%) ng/mL, respectively. Extraction procedure substitutions decreased assayed sirolimus from 13.63 to 4.60 (-66.25%) and 8.07 (-40.79%) ng/mL for cyclosporine and tacrolimus. Cyclosporine concentration increased from 274.60 to 391.30 (+42.50%) ng/mL using sirolimus extraction reagents and to 757.30 (+175.78%) ng/mL using tacrolimus extraction reagents. Cross-reactivity was observed between the tacrolimus assay and sirolimus and cyclosporine extraction reagents. Significant changes, both positive and negative, are observed in assayed drug concentration when incorrect extraction procedures are used in the Abbott i2000 tacrolimus, sirolimus, and cyclosporine assays. Preanalytic extraction procedures should be investigated when performing root cause analysis for erroneous therapeutic drug values.

Examination and comparison of the RNA extraction methods using mouse serum.

Serum microRNAs (miRNAs) are considered useful as non-invasive biomarkers for different diseases. However, the optimal method for extracting RNAs from serum is currently unknown. In the present study, several RNA extraction kits were used to examine the optimal kit. RNAs were extracted from the serum of 8-week-old C57BL/6NJcl male mice following the protocol of each RNA extraction kit. The yield of the extracted RNA samples was calculated, and an Agilent Bioanalyzer was used to assess the electrophoretic patterns. An Agilent mouse miRNA microarray was utilized to confirm the expression patterns of the extracted RNA samples. The results revealed significant differences in RNA yields from the miRNeasy Serum/Plasma Advanced kit and mirVana™ PARIS™ RNA and Native Protein Purification Kit compared with almost all other samples. Further, two peaks were determined in the miRNeasy Serum/Plasma Advanced kit using a small RNAs kit of Agilent Bioanalyzer, including one at 20-40 nucleotides (nt) and another at ~40-100 nt, whereas the other reagents had a single peak. This revealed that the extracted RNAs may differ in composition based on the RNA extraction method. Some types of miRNAs were only detected with certain RNA extraction reagents. This suggested that different RNA extraction reagents may cause differences in the types of miRNAs detected. On the other hand, the miRNAs commonly expressed by the three RNA extraction reagents are highly correlated in expression levels.