Simple Dilution Research Articles

Plasmatic and urinary pyroglutamic acid measurement by capillary zone electrophoresis in chronic poisoning with acetaminophen in children

An increase of pyroglutamic acid, or 5-oxoproline plasmatic concentration was reported in metabolic acidosis observed after chronic intake of some drugs, as acetaminophen. We developed a simple, fast and reproducible method by capillary zone electrophoresis using a commercial Anion Analysis Kit® to quantify pyroglutamic acid, in plasma after acetonitrile precipitation, and after simple dilution in urines. Fumaric acid was used as internal standard in both. In less than 7min, the method separates pyroglutamic acid from other organic and inorganic anions. The method is linear between 0.25 and 10mmol/L in plasma, and 0.15 and 10mmol/L in urines. The quantification limits are 0.25mmol/L and 0.15mmol/L for plasma and urines, respectively. For repeatability and intermediate precision, the variation coefficients are less than 15% and the bias values are between ±10%. For the 2 matrices, the recoveries are between 88% and 101%. The method does not interfere with physiological organic and inorganic anions. Pyroglutamic acid concentrations measured in 9children were between 0.45 and 3.96mmol/L in the plasma and between 0.15 and 3.2mmol/L in the urine. No correlation between pyroglutamic acid and acetaminophen concentrations were found, regardless of the biological media. In conclusion, our method measures pathophysiological concentrations of pyroglutamic acid and highlights the increase in other organic acids that may explain metabolic acidosis due to chronic acetaminophen intake.

A Simple Dilution Method for Preparation of Different Aggregates from Oleic Acid/CHAPSO Bicelles.

We evaluated the effect of dilution on both the size and packing density of aggregates prepared from a fatty acid (oleic acid, OA)/detergent (3-[(3-cholamidopropyl)dimethylammonio]-2-hydroxypropane sulfonate (CHAPSO)) bicelle as a parent for functional membrane materials. The sizes of the aggregates formed at different molar ratios, XOA(= [OA]/([OA]+[CHAPSO])), of 0.3 and 0.7 and their parent bicelles were measured by dynamic light scattering and transmission electron microscopy; their packing density was evaluated by deconvolution of the fluorescence spectrum, where Laurdan molecules were used as a probe. The experimental results showed that the bicelles formed aggregates upon dilution because of the hydration of CHAPSO. The packing density of the nano-ordered aggregate formed at XOA = 0.3 was much greater than that of the aggregate formed at XOA = 0.7, implying the formation of an ordered aggregate under the condition of XOA = 0.3.

Microstructure transition gradients in titanium dissimilar alloy (Ti-5Al-5V-5Mo-3Cr/Ti-6Al-4V) tailored wire-arc additively manufactured components

The nature of the chemical mixing and microstructure gradients that occur across the interface transition, when manufacturing tailored components with the two high-performance dissimilar titanium alloys (Ti-6Al-4V (Ti-64) and Ti-5Al-5V-5Mo-3Cr (Ti-5553)) by the wire-arc additive manufacturing (WAAM) process, are reported. It has been shown that a relatively long-range chemical gradient occurs during the transition between layers produced with the two different titanium alloys, due to convective mixing in the melt pool between the substrate layers and new alloy wire. This resulted in a stepwise exponential decay composition profile normal to the layers, the width of which can be described by a simple dilution law, with steep local composition gradients seen within the boundary layers at the fusion boundary of each individual layer. The alloy-alloy composition gradients had little effect on the β-grain structure. However, they strongly influenced the transformation microstructure, due to their effect on the parent β-phase stability and the β → α transformation kinetics and reaction sequence. The microstructure gradient seen on transitioning from Ti-64 → Ti-5553 was significantly more abrupt, compared to when depositing the two alloys in the reverse order. Under WAAM thermal conditions, Ti-64 appears to be more sensitive to the effect of adding β-stabilising elements than when Ti-5553 is diluted by Ti-64, because at high cooling rates, stabilisation of the β phase readily suppresses α nucleation when cooling through the β transus, and the normal Ti-64 lamellar transformation microstructure is abruptly replaced by finer scale α laths generated by precipitation during subsequent reheating cycles.

Targeted profiling of 24 sulfated and non-sulfated bile acids in urine using two-dimensional isotope dilution UHPLC-MS/MS.

Bile acids serve as biomarkers for liver function and are indicators for cholestatic and hepatobiliary diseases like hepatitis, cirrhosis, and intrahepatic cholestasis of pregnancy (ICP). Sulfation and renal excretion of bile acids are important elimination steps. The power of ultra high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) allows specific profiling of primary and secondary bile acids as well as their sulfated counterparts. Twenty-four sulfated and non-sulfated primary and secondary bile acids were quantified in urine with 15 corresponding stable isotope labeled internal standards by using two-dimensional UHPLC-MS/MS. The sample preparation was based on a simple dilution with a methanolic zinc sulfate solution followed by an automated online solid phase extraction clean up. The validation results of the method fulfilled the criteria of the European Medicine Agency (EMA) "Guideline on bioanalytical method validation". To verify fitness for purpose, 40 urine samples were analyzed which showed an average of 86% sulfation, 9.1% taurine-conjugation, 14% non-conjugation, and 77% glycine-conjugation rates. Lossless one-pot sample preparation, automated sample purification, and high number of internal standards are major innovations of the presented profiling method, which may allow diagnostic application of BA profiling in the future.

Simple and fast batch injection analysis method for monitoring diuron herbicide residues in juice and tap water samples using reduced graphene oxide sensor

In this paper, a new analytical method was developed using batch injection analysis with amperometric detection and reduced graphene oxide modified glassy carbon (GCE/rGO) electrode for the simple, fast, sensitive, and effective diuron (DIU) herbicide determination in tap water and food beverages (whole juice of grape and orange) samples. The GCE/rGO sensor provided a 7.6-fold increase in the DIU electrochemical oxidation current compared to the bare electrode, which resulted in a method with good sensitivity and detectability. The developed method provided important analytical characteristics for the DIU quantification, such as detection limit of 0.36 μmol L−1 (84 μg L-1) below the maximum residual limit of U.S Environmental Agency, linear range from 5 to 50 μmol L-1, good precision (RSD < 3.7 %), and accuracy (recovery levels ranging from 80.8–105.5 %). Furthermore, the minimal sample preparation (simple dilution in the supporting electrolyte) associated with a high analytical frequency (150 analyses per hour) stands out. In this sense, the proposed method proves to be an alternative analytical tool for applications in the environmental and food interest samples analysis.

Simultaneous Analysis of the Metabolome and Lipidome Using Polarity Partition Two-Dimensional Liquid Chromatography-Mass Spectrometry.

Comprehensive metabolic profiling is a considerable challenge for systems biology since the metabolites in biological samples have significant polarity differences. A heart-cutting two-dimensional liquid chromatography-mass spectrometry (2D-LC-MS) method-based polarity partition was established to analyze both the metabolome and lipidome in a single run. Based on the polarity partition strategy, metabolites with high polarity were retained and separated by one-dimensional hydrophilic chromatography, while low- and medium-polarity lipids were collected into a sample loop and injected into two-dimensional reversed-phase chromatography for separation. A simple online dilution strategy realized the online coupling of the 2D-LC-MS, which effectively solved band broadening and peak distortion caused by solvent incompatibility. Moreover, a dual gradient elution procedure was introduced to further broaden the coverage of low-polarity lipids. The metabolites' log P values, which this 2D-LC-MS method could analyze, ranged from -8.79 to 26.86. The feasibility of the 2D-LC-MS system was demonstrated by simultaneous analysis of the metabolome and lipidome in rat plasma related to depression. A total of 319 metabolites were determined within 40 min, including organic acids, nucleosides, carbohydrate derivatives, amino acids, lipids, and other organic compounds. Finally, 44 depression-related differential metabolites were screened. Compared with conventional LC-MS-based methods, the 2D-LC method covered over 99% of features obtained by two conventional methods. In addition, the selectivity and resolution of the hydrophilic metabolites were improved, and the matrix effects of the hydrophobic metabolites were reduced in the developed method. The results indicated that the established 2D-LC system is a powerful tool for comprehensive metabolomics studies.

Determination of Benzoic Acid and Benzyl Alcohol in E-Liquids (JUUL™ Pods) by Isotopic Dilution High-Performance Liquid Chromatography and Tandem Mass Spectrometry.

The new pod devices like JUUL™, Vuse Alto™, myblu™, and other ?pod-mod" related products had a huge impact on the e-cigarette market, especially among teens and young adults due in particular to aggressive marketing on social media, wide availability, and discrete use due to their special design. These pod devices are designed to deliver nicotine levels per puff comparable to combustible cigarettes while producing smaller amounts of visible exhaled aerosol from the heating of e-liquids. Some of these liquids contain high concentrations of acids, such as benzoic acid, to allow higher nicotine deliveries with less harshness and throat irritation. Benzoic acid is a potential source of the human carcinogen benzene and a chemical of concern. Besides acids, flavoring agents such as benzyl alcohol, a local anesthetic that could facilitate tobacco smoke inhalation are also common in these devices. Both benzoic acid and benzyl alcohol in e-liquids might be of relevance for the health risk of vapers. An isotope dilution high-performance liquid chromatography-tandem mass spectrometry (ID-HPLC-MS/MS) method has been developed for the detection of benzoic acid and benzyl alcohol in the JUUL™ brand e-liquids. The sample preparation consisted of a simple dilution followed by a mechanical stirring process. ID-HPLC-MS/MS was used to separate, identify, and quantify the benzoic acid and/or benzyl alcohol in diluted extracts. Detection limits were 0.11 and 9.05 ng/μL for benzyl alcohol and benzoic acid, respectively. Product variability estimated from the analysis of seven different e-liquids in triplicates (n = 21) yielded relative standard deviations ranging from 4.3% to 16.0% for benzyl alcohol and 6.3% to 11.1% for benzoic acid. The amount of benzoic acid (32.8 ± 2.8 mg/g; 3.3 ± 0.3%, w/w) and the nicotine-benzoic acid molar ratio (1.15 ± 0.02) remained relatively consistent among pod flavors. [Contrib. Tob. Nicotine Res. 30 (2021) 212-220].

Pseudophase microextraction for in-line sample concentration in micellar electrokinetic chromatography

Pseudophase microextraction (PPME) as a simple in-line sample concentration technique in micellar electrokinetic chromatography (MEKC) is presented. In contrast to popular electric-field driven stacking techniques in MEKC such as sweeping, PPME is pressure-driven. The technique afforded up to 403-2968x improvements in peak heights for fenoprop, dichlorprop, 1- and 2-naphthol compared to typical injection. Under the same MEKC conditions, the improvements in PPME were up to 23-59x better compared to sweeping. Briefly in PPME, the entire capillary was loaded (up to 20 capillary volumes) with the sample prepared in a dilute solution of cetyltrimethylammonium bromide ([CTAB] > critical surface aggregation concentration). The CTAB formed aggregates at the inner capillary walls and these aggregates acted as a stationary chromatographic pseudophase. After clean-up via flushing the capillary with purified water, the MEKC background solution (BGS) with sodium dodecyl sulfate was then introduced by pressure from the outlet end to elute the retained analytes. The analytes concentrate at front of the BGS and the front was moved to the inlet end of the capillary prior to MEKC. Optimization strategies and current limitations in PPME-MEKC are described. The linear ranges using a 4 capillary volume sample load obtained for fenoprop, dichlorprop, 1- and 2-naphthol were between 1 and 160 ng/mL (r2s ≥ 0.996), LOQs = 1–2.5 ng/mL and repeatability %RSDs (n = 6) were ≤5% (intra-day) and ≤7% (inter-day) (using low analyte concentrations 1-5x LOQ). PPME-MEKC with simple dilution of fortified real samples (no off-line sample concentration) was also able to detect low levels of dichlorprop (10 ng/mL, limit set in Australia) and 1- and 2−naphthol (7.5–15 ng/mL) in a drinking water and natural water sample, respectively (% recovery = 84–108%). The concept of PPME may find use in other modes of capillary electrophoresis and other nano-microscale separations.

Calcium monofluoride as a target molecule for determination of organic fluorine in gasoline and its components using high-resolution continuum source flame molecular absorption spectrometry

High-resolution continuum source molecular absorption spectrometry (HR-CS MAS) is widely applied to determine fluorine in various materials, and its graphite furnace option with calcium monofluoride as a target molecule is popular. However, up to now, the idea of using the molecule in flame molecular absorption spectrometry seemed impossible or impractical due to the expected low sensitivity and detectability. The following paper completely changes this view. The CaF molecule has been proven to be an excellent tool for determining organic fluorine in gasoline using flame molecular absorption spectrometry, giving benefits over previously used gallium monofluoride. The following favourable circumstances decide about it: 1) relatively high population of CaF molecules in preferable air-acetylene flame, 2) possibility of using oil calcium “standard” as a source of calcium, which is well dissoluble in both xylene (solvent) and gasoline, 3) extremely good stability of baseline in the used spectrum range, 4) relatively high freedom from spectral and chemical interferences and 5) possibility of summing of many pixels response. The analysis relies on simple sample dilution in xylene, with the addition of oil calcium “standard” to obtain 1500 mg L−1 Ca in measured solutions. Simplified standard addition calibration is used to overcome the effect of variability of samples (various physical properties and combustion characteristics). The signals of four CaF rotational “lines” are summed. The only undesirable spectral effect is a molecular absorption at the second “line” (606.0368 nm), which is attributed to the absorption by the CaOH molecule formed in the flame. The effect can be easily overcome by using the least square background correction, LSBC. The characteristic concentration and the detection limit for solutions are 6 and 0.6 mg L−1, respectively. The detection limit recalculated for the original sample is 2 mg kg−1 in the case of alkalyte, reformate and gasoline, while it is 6 mg kg−1 in the case of volatile isomerizate, mainly due to necessary higher sample dilution. The calibration curve is linear over at least two orders of magnitude. Heptafluorobutanol has been found a suitable standard. The agreement of the results of the proposed method and standard combustion ion chromatography is satisfactory.

Evaluation of extraction-free RT-PCR methods for faster and cheaper detection of SARS-CoV-2 using two commercial systems

ObjectiveWhen using high-throughput batched diagnostic platforms based on RT-PCR for SARS-CoV-2 detection, avoidance of the conventional nucleic acid extraction step can help to reduce the turnaround time and increase processivity. This approach can also spare reagents and plasticware, which have experienced a shortage during the initial waves of the pandemic, reducing the overall testing costs. MethodsThis study evaluated the performance of extraction-free protocols based on simple dilution of the specimen in sterile RNAse free water (with or without a heating step) in comparison to standard RNA extraction protocols, using two commercial kits for molecular detection of SARS-CoV-2 (Allplex™ SARS-CoV-2 assay and Allplex™ SARS-CoV-2/FluA/FluB/RSV assay) in nasopharyngeal swabs (NPS). ResultsCompared with conventional protocols, extraction-free protocols based on sample dilution without a heating step exhibited a lower analytical sensitivity: 74.0% and 82.1% with the Allplex™ SARS-CoV-2 assay (tested with 139 NPS samples) and the Allplex™ SARS-CoV-2/FluA/FluB/RSV assay (tested with 69 NPS samples), with a mean increase of Ct values of +2.04 and +1.32, respectively. Most false negative results were observed with sampled low viral load. Including a step of heat exposure did not improve but actually decreased the analytical sensitivity of the assay. ConclusionsResults confirmed that extraction-free protocols could be a faster and cheaper approach to SARS-CoV-2 detection in NPS samples, which could improve processivity of diagnostic platforms.

Common Pitfalls and Recommendations for Using a Turbidity Assay to Study Protein Phase Separation.

The turbidity assay is commonly exploited to study protein liquid-to-liquid phase separation (LLPS) or liquid-to-solid phase separation (LSPS) processes in biochemical analyses. Herein, we present common pitfalls of this assay caused by exceeding the detection linear range. We showed that aggregated proteins of high concentration and large particle size can lead to inaccurate quantification in multiple applications, including the optical density measurement, the thermal shift assay, and the dynamic light scattering experiment. Finally, we demonstrated that a simple sample dilution of insoluble aggregated protein (LSPS) samples or direct imaging of liquid droplets (LLPS) can address these issues and improve the accuracy of the turbidity assay.

Development, validation, and application of an LC–MS/MS method for the quantification of the novel antibiotic drug lefamulin (Xenleta®) and its main metabolite 2R-hydroxy lefamulin in human plasma

Lefamulin (Xenleta®) is a first in-class systemic pleuromutilin antibiotic that inhibits bacterial protein synthesis and selectively binds to a highly conserved region of the peptidyl transferase center of the bacterial 50S ribosomal subunit. A total of twenty-five Phase 1 clinical studies, one Phase 2 study in acute bacterial skin and skin structure infections (ABSSSI), and two pivotal Phase 3 studies in adults with community acquired bacterial pneumonia (CABP) have been completed. Xenleta® (lefamulin) has been approved by the FDA on August 19, 2019, by Health Canada on July 10, 2020, and by the EMA on July 28, 2020 for the oral and IV treatment of CABP in adults.For and during the clinical development, simple, sensitive, precise, and selective LC–MS/MS methods were developed and validated, first for lefamulin alone and later for the simultaneous quantification of lefamulin and its main metabolite 2R-hydroxy lefamulin in human plasma.Chromatographic separation in the current method was achieved on a reverse phase C18 column using gradient elution at a flowrate of 500 μL/min consisting of a mobile phase of water (A) and methanol (B) each containing 0.1 % formic acid (v/v) with a run time of 8.0 min. The detection and quantification of the analytes were performed on AB Sciex Triple Quad 5500 using multiple reaction monitoring operated in positive electrospray ionization mode after a simple plasma protein precipitation cleanup and dilution. The method was linear over the concentration range of 1.00−1 000 ng/mL (r ≥ 0.999) for lefamulin und 1.00–500 ng/mL (r ≥ 0.999) for 2R-hydroxy lefamulin. No significant matrix effects and a good extraction recovery were observed. The within- and between-run precision and accuracy were within the acceptable limits, and both analytes were found to be stable throughout the short term, long term, and freeze thaw stability studies. This current validated method was successfully applied in five Phase 1 and two Phase 3 studies.

Clinimetallomics: Arsenic Speciation in Urine from Patients With Arsenism by HPLC-ICP-MS

Clinimetallomics is proposed as a branch of metallomics that focuses on the study of the metallome in clinical samples of urine, blood, and tissues. As the clinical diagnosis of arsenic poisoning is mainly based on the concentration of total arsenic in urine, the toxicity of arsenic varies greatly in different speciation. Analysis of arsenic speciation with excessive total arsenic in urine can provide a basis for precise treatment. It can also be used to understand the fate of arsenic in the body of patients with arsenic poisoning after treatment with sodium dimercaptopropane sulfonate. In this study, a HPLC-ICP-MS method was established for the determination of arsenic species in urine samples from patients diagnosed with arsenism. Use the established method to detect urine samples, which can be directly assayed after simple sample dilution with 20 mmol/L EDTA-2Na. With the concentration of arsenic speciation in the range of 1.0~100.0 ng/mL, the linear correlation coefficient was higher than 0.99996. The recoveries were between 92.4% and 109.0%. The precision of the concentration and retention time (n = 3) were less than 3.0% and 0.3%, respectively, and the detection limit was between 1.42 ng/mL and 1.86 ng/mL. This method can be applied to arsenic speciation in the urine of healthy people, in patients treated for arsenic poisoning, and in patients diagnosed with arsenism.

Quadruple-Checkerboard: A Modification of the Three-Dimensional Checkerboard for Studying Drug Combinations.

The concept of drug-combination therapy is becoming very important mainly with the drastic increase in resistance to drugs. The Quadruplecheckerboard, also called the Q-checkerboard, aims at maximizing the number of possible combinations that can be obtained between four drugs in one experiment to minimize the time and work needed to accomplish the same results with other protocols. This protocol is based on the simple micro dilution technique where the drugs are diluted and combined together in several 96-well plates. In the first set of 96-well plates, Muller-Hinton broth is added followed by the first required drug (e.g., Cefotaxime here) to serially dilute it. After the first step is done, another set of 96-well plates is used to dilute the second drug (e.g., Amikaci), which will be transferred by removing a specific volume of drug 2 and put in the corresponding wells in the first set of 96-well plates that contains drug one. The third step is done by adding the required concentrations of the third drug (e.g., Levofloxacin), to the appropriate plates in the initial set containing combination of drug 1 and 2. The fourth step is done by adding the required concentrations of the fourth drug (e.g., Trimethoprim-sulfamethoxazol) into the appropriate plates in the first set. Then, E. coli ESBL bacterial inoculum will be prepared and added. This method is important to evaluate all the possible combinations and has a wider range of possibilities to be tested furthermore for in vivo testing. Despite being a tiring technique requiring a lot of focus, the results are remarkable and time saving where a lot of combinations can be tested in a single experiment.

Development of ELISA formats for polymyxin B monitoring in serum of critically ill patients

Treating infections in critically ill patients often requires the use of last-line antibacterial drugs such as polymyxins with a narrow therapeutic window and high toxicity. In critically ill patients, the drug pharmacokinetics changes significantly, and as a result, the antibiotic concentrations in blood and infection foci become suboptimal, which leads to therapeutic failures or toxic manifestations. For timely dosage adjustments, a competitive ELISA-based method using antibodies to polymyxin В (PMB) was developed. Among the several considered assays, a direct antibody-coated format was selected for its short duration (1.5 h) and the best agreement with the LC–MS/MS data (R2 = 98 %). The assay dynamic measurement range (IC20-IC80) could be substantially shifted by changing the ratio of immunoreagents. To conveniently measure the therapeutic range of PMB concentrations, it was adjusted to 5.0–192 ng/mL, allowing the samples to be analyzed after a simple 100-fold dilution with the assay buffer. The ELISA sensitivity expressed in half-inhibition concentration (IC50) and the limit of detection were 30.6 and 1.8 ng/mL, respectively. The assay cross-reactivity towards the related analogue colistin (COL) was 95 %, and this compound could also be adequately quantified by the same assay. The PMB and COL recovery from the spiked serum samples was similar and constituted 98–109 %. The trial drug monitoring was carried out in 3 patients with Gram-negative sepsis, and the established pharmacokinetic profiles of PMB revealed the necessity for individual dosage adjustment.

Rapid, label-free, and sensitive point-of-care testing of anti-SARS-CoV-2 IgM/IgG using all-fiber Fresnel reflection microfluidic biosensor.

Graphical abstractThe ongoing global pandemic of SARS-CoV-2 has promoted to develop novel serological testing technologies since they can be effectively complementary to RT-PCR. Here, a new all-fiber Fresnel reflection microfluidic biosensor (FRMB) was constructed through combining all-fiber optical system, microfluidic chip, and multimode fiber bio-probe. The transmission of the incident light and the collection and transmission of Fresnel reflection light are achieved using a single-multi-mode fiber optic coupler (SMFC) without any other optical separation elements. This compact design greatly simplifies the whole system structure and improves light transmission efficiency, which makes it suitable for the label-free, sensitive, and easy-to-use point-of-care testing (POCT) of targets in nanoliter samples. Based on Fresnel reflection mechanism and immunoassay principle, both the SARS-CoV-2 IgM and IgG antibodies against the SARS-CoV-2 spike protein could be sensitively quantified in 7 min using the secondary antibodies-modified multimode fiber bio-probe. The FRMB performs in one-step, is accurate, label-free, and sensitive in situ/on-site detection of SARS-CoV-2 IgM or IgG in serum with simple dilution only. The limits of detection of SARS-CoV-2 IgM and SARS-CoV-2 IgG were 0.82 ng/mL and 0.45 ng/mL, respectively. Based on our proposed theory, the affinity constants of SARS-CoV-2 IgM or IgG antibody and their respective secondary antibodies were also determined. The FRMB can be readily extended as a universal platform for the label-free, rapid, and sensitive in situ/on-site measurement of other biomarkers and the investigation of biomolecular interaction. Supplementary InformationThe online version contains supplementary material available at 10.1007/s00604-021-04911-0.

Development and validation of an LC-HRMS method for the determination of pyrrolizidine alkaloids and quinolones in honey employing a simple alkaline sample dilution

Development and validation of an LC-HRMS method for the determination of pyrrolizidine alkaloids and quinolones in honey employing a simple alkaline sample dilution

A stable and sensitive enzyme-linked immunosorbent assay (ELISA) for the determination of metsulfuron-methyl residues in foods.

A hapten of metsulfuron-methyl was successfully designed and synthesized from 2-methylester-phenylsulfonamide and succinic anhydride, and the polyclonal antibody against metsulfuron-methyl was prepared by immunization procedure with the hapten-bovine serum albumin conjugate. A stable and sensitive direct competitive enzyme-linked immunosorbent assay (dcELISA) method had been developed under the optimal conditions. The sensitivity (IC50 ) was 37.03± 1.87µg/L, and the detection line (IC15 ) was 1.57± 0.11µg/L. Rice, wheat, oat, flaxseed, milk, and water were chosen to study the recovery test and the recovery rates were 83.11%-117.44% . The matrix effect was eliminated by a simple dilution of the sample extracts. The results from dcELISA were well agreement with the results from HPLC-MS. It was indicated that the developed method had good accuracy and stability. It could be applied for the detection of metsulfuron-methyl residues. It was worth mentioning that the antibody could recognize metsulfuron-methyl and tribenuron-methyl with cross-reactivities of 100% and 49.72%, respectively. In order to understand the cross-reactivity, molecular modeling including molecular alignment and electrostatic potential surfaces were introduced. It was found that the special group of metsulfuron-methyl played an important role, especially on C3 position of the phenyl group. PRACTICAL APPLICATION: A stable, sensitive, and low-cost dc ELISA method had been developed with good accuracy and applied in the determination of metsulfuron-methyl in foods. Molecular simulation was introduced to understand the specificity between the antibody and the analyst. It was a good method to study the cross-reactivity between the antibody and the analyst or analogue.

Antimicrobial activity of Hylocereus undatus (White dragon fruit) peel extract using ethanol against Methicillin-resistant Staphylococcus aureus

The worldwide development of multi-drug resistant microbial strain is progressively restricting the efficacy of the latest drugs and essentially leads to failure in the treatment of infection. Hylocereus undatus (White dragon fruit) peel extract possesses phytochemicals and antimicrobial properties, namely flavonoid, alkaloids, phenols, and carbohydrate, etc. In this study the antimicrobial activity of ethanolic extract of Hylocereus undatus peel against Methicillin-resistant Staphylococcus aureus was evaluated using disc diffusion and simple dilution. The result showed that the Hylocereus undatus (White Dragon Fruit) extract inhibits the growth of bacteria in the range of 6mm. The results in the disc diffusion method of Hylocereus undatus (White dragon fruit) peel extract showed that there's no significant difference in the effectiveness of antimicrobial components of extracted Hylocereus undatus (White Dragon fruit) peel against Methicillin-Resistant Staphylococcus aureus by the zone of inhibition using ethanol. Thus, these findings could be used further to understand the antimicrobial property of Hylocereus undatus (White dragon fruit) peel.

An isotope dilution liquid chromatography-tandem mass spectrometry candidate reference measurement procedure for aldosterone measurement in human plasma.

Accurate quantitation of aldosterone is clinically important in standardized testing for primary aldosteronism. The results are often variable when performed by clinical immunoassays. To standardize and ensure the accuracy of clinical systems, reference measurement procedures (RMPs) with higher metrological order are required. A simple and reliable isotope dilution LC-IDMS/MS-based measurement procedure for human plasma aldosterone has been developed. This method involved plasma spiked with a deuterium-labelled internal standard, equilibrated for 0.5h, and extracted by liquid-liquid extraction (LLE) without derivatization. Aldosterone and its structural analogues were baseline separated with a C18-packed UHPLC column with gradient elution within 7min. The signal intensity variability and measurement imprecision were reduced by bracketing calibration during plasma aldosterone value assignment. The limit of detection (LoD) was 19.4pmol/L with a signal-to-noise ratio (S/N) > 3. The lowest limit of quantification (LLoQ) was 27.7pmol/L (S/N > 10 and CV < 10.0%). LLE was performed with 1mL of n-hexane/ethyl acetate (3:2, v/v), and the extraction recovery was determined to be 92.15 ± 3.54%. The imprecisions were ≤ 3.18% for samples at 124.8, 867.0, and 2628.5pmol/L. The recoveries were 98.11-101.61%. The relative bias between this candidate RMP and the established RMP was 2.76-1.89%. The linearity response ranged from 27.7 to 2774.4pmol/L with R2 = 0.999. The method performance met the requirements of RMPs (≤ 5% total CV and ≤ 3% bias). Furthermore, the developed method was applied to evaluate immunoassays through 41 patient sample comparisons. The calibration and measurement capability (CMC) of this method were also evaluated by measuring these samples. The candidate RMP can serve as an accurate reference baseline for routine methods and can be used for value assignment for reference materials. Selected ion chromatograms by LC-MS/MS using a C18 column for aldosterone and its structural analogues.