Standard Method For Determination Research Articles

Effects of Temperature and pH on the Stability of Protease Produced by Alcaligenes faecalis P2

Background: Bacterial proteases represent a group of very important industrial enzymes. They are involved in the hydrolysis of peptide bond found in protein. Industrial application of bacterial proteases has been limited by low yield and instability at biotechnological process conditions. This study was design to investigate the effect of temperature and pH on the stability of protease produced by Alcaligene faecalis strain P2.
 Methodology: Protease-producing bacteria were isolated from beans effluent-impacted soil and screened for protease production on Casein agar plate. Protease assay was carried out following standard method for protease determination and the stability of the protease produced was investigated over temperature range of 20 to 90 oC and pH range of 3 to 12. The protease-producing bacterium was identified using its molecular characteristics.
 Results: Protease assay result showed that the amount of tyrosine released by one unit of the crude enzyme was 0.176 µmol/mL resulting in 0.19 U/mL protease activity. Stability studies showed that the protease had wide spectrum of pH and temperature stability. The protease was most stable at pH 9 and temperature of 40 oC after 30 min. Maximum protease activity of 1.259 U/mL was recorded with pH 9 after 30 min. The protease-producing bacterium was classified as Alcaligene faecalis P2 based on phylogenetic analysis of its 16S gene analysis. The sequences have been submitted to GenBank under the accession number MZ477004.
 Conclusion: This study therefore has demonstrated that beans effluent-impacted soil harbours protease producing bacteria with high industrial potentials. In addition, the study revealed that the protease produced in this study can retain its activity over wide temperature and pH.

Determination of the total acid number of Brazilian crude oil samples: Theoretical and experimental evaluation of three standard methods

The total acid number (TAN) is commonly used to assess the quality of crude oil, petroleum products, lubricants, biodiesel, and biodiesel blends and can be determined using several standard methods. The validation of these methods usually involves the estimation of their precision, which is related to repeatability and reproducibility. Herein, we theoretically compare the performances of potentiometric (ASTM D664 and ABNT NBR 14448) and thermometric (ASTM D8045) standard methods of TAN determination in terms of sample type scope, operational TAN range, precision, and the effects of interferents. In addition, these methods are used to experimentally determine the TANs of four Brazilian crude oil samples (API gravity = 13.2–31.0), and the precision of these values is found to be affected by sample solubility, sample size, and hydrolysable salt type and content. Thus, our work provides valuable reference information and facilitates the selection of optimal TAN determination methods, which should ultimately help minimize the adverse environmental and financial effects of inaccurate TAN determination in the oil refining and production chain.

RILEM Standard: testing methods for determination of the double-K criterion for crack propagation in concrete using wedge-splitting tests and three-point bending beam tests, recommendation of RILEM TC265-TDK

In this recommendation, standard testing methods for determination of the double-K criterion for Mode I crack propagation in concrete using wedge-splitting tests and three-point bending beam tests are specified for the fracture parameters of the initial cracking toughness K_{Ic}^{ini} and the unstable fracture toughness K_{Ic}^{un}. Along with the recommendation of the standard testing methods, the theoretical background of the double-K criterion, the calculation methods and the results of round-robin testing for determining the double-K fracture parameters are presented in technical reports [1, 2]. The recommendation of the standard testing methods includes geometry for specimens, fabrication of specimens, testing machine, load transmission system and supports, measuring instruments, determination of initial cracking load Pini, determination of measured maximum load Pmax and initial compliance ci, calculation methods for wedge-splitting test and three-point bending beam test, as well as the testing results and testing report forms. According to these methods, the measured fracture parameters of double-K criterion can be used to describe the onset of cracking, and the onset of unstable cracking or failure for predicting crack initiation, structure failure and crack stability in concrete structures.

New Approach to Concurrent VS and VP Measurements Using Bender Elements

Abstract Bender elements (BEs) have become a routine geotechnical laboratory tool for seismic wave velocity measurements. Since the 1980s, this testing technique has gained popularity, currently being available in many geotechnical laboratories worldwide in a variety of apparatuses. The advantage of simultaneously measuring small- and large-strain soil stiffnesses in each device and the easiness and low-cost implementation of BE are the main reasons for their common application. Although there is already a standardized procedure for BE testing (ASTM D8295-19, Standard Test Method for Determination of Shear Wave Velocity and Initial Shear Modulus in Soil Specimens Using Bender Elements), the use of high-frequency pulses for the simultaneous measurement of compressional (VP) and shear (VS) wave velocities is not considered. In contrast, the use of high-excitation frequencies is usually discouraged, as they tend to induce spurious participation of high-vibration modes in the BE response. However, this work shows that the use of higher vibration modes can be advantageous to evaluate P-wave velocities from standard BE testing. Thus, this paper presents a new approach for the concurrent measurements of VP and VS using a typical installation of BE. The new approach is first demonstrated by experimental measurements and subsequently validated using high-frequency laser vibrometer measurements of the actual BE deformation (nanometer scale) under different excitation frequencies. The laser vibrometer measurements show the displacements of the transmitter BE as a function of the input frequency in not only the horizontal but also in the vertical directions, demonstrating the generation of P-waves when higher vibration modes are excited. The measured VP values are shown to be in good agreement with the predicted values using Biot’s equations. Thus, the proposed methodology addresses the current knowledge gap in the use of BE for concurrent P-wave and S-wave velocity measurements. The generated wavelengths are large enough to travel through the soil skeleton instead of the pore water only.

Evaluation of Current ASTM Standards for ASR Prevention When Fine Lightweight Aggregates Are Used

Abstract Previous research has shown that using fine lightweight aggregate (FLWA) can be a promising strategy to mitigate alkali-silica reaction (ASR) in mortar and concrete. However, limited studies focused on assessing current ASTM standards for their applicability in evaluating the efficacy of ASR mitigation using FLWAs. In this study, three commercially used FLWAs (expanded shale, clay, and slate) were investigated in mortar and concrete mixtures with reactive aggregates of different levels of reactivity. ASTM test methods, including ASTM C289-07, Standard Test Method for Potential Alkali-Silica Reactivity of Aggregates (Chemical Method) (Withdrawn 2016); ASTM C1260-14, Standard Test Method for Potential Alkali Reactivity of Aggregates (Mortar-Bar Method) (Superseded); and ASTM C1293-18, Standard Test Method for Determination of Length Change of Concrete due to Alkali-Silica Reaction (Superseded), were completed. Fine normal-weight aggregates were replaced by the FLWA at 25 % and 50 % by volume in the concrete mixtures, and 25 %, 50 %, and 100 % in mortar mixtures. Results showed that ASTM C1260-14 and ASTM C1293-18 can be used to evaluate the mitigation efficacy when pre-wetted FLWAs were used. The ASTM C289-07 test is not a reliable test method to study the reactivity of the FLWAs, but the results can be used to indicate the alkali-consuming ability of the FLWAs. All three FLWAs were effective in reducing ASR-induced expansion in both ASTM C1260-14 and ASTM C1293-18. The investigated FLWAs were especially effective in the concrete when moderately reactive aggregates, as classified by ASTM C1778-14, Standard Guide for Reducing the Risk of Deleterious Alkali-Aggregate Reaction in Concrete (Superseded), were used. For concrete with a highly reactive aggregate or very highly reactive aggregate, other mitigation strategies may need to be combined with FLWAs to effectively mitigate ASR.

Efficiency Determination of Compressor Embedded Induction Motors

Compressors operating in refrigeration systems, typically with built-in induction motors, represent an important energy demand. The applicability of standard methods for efficiency determination of induction motors is limited in this context by the constructive characteristics of these motors, such as the shared compressor frame and the inaccessibility of the shaft. The main contributions of this paper are to provide an overview of standard and alternative methods for efficiency testing of single and polyphase motors, according to technical standards and to the state of the art, and to offer guidelines for the method selection. The options cover dynamometer tests, loss segregation and equivalent circuit methods, some even applicable without direct access to the motor. Experimental application examples in single and three- phase motors are presented. Beyond the context of compressors, the discussion is relevant for inaccessible motors in general.

A Recommendation for a Pre-Standardized Marine Microalgal Dry Weight Determination Protocol for Laboratory Scale Culture Using Ammonium Formate as a Washing Agent.

Simple SummaryMicroalgae are increasingly recognized as a source of valuable biomass with numerous health benefits. Cleaning of marine microalgal biomass is very crucial for microalgal studies as the salt on the microalgae cells will lead to overestimation of biomass determination. Incomplete washing of salt from microalgae could also interfere with the nutritional analyses. The biomass, especially dry weight, has been utilized for nutritional or compositional evaluation. Although standard methods of marine microalgal dry weight determination are available, these methods did not provide comprehensive details, and the parameters vary among themselves. Without a standard method, a comparison of results among previous studies can be misleading and unreliable. Therefore, the current study aimed to investigate and determine the ideal setting of several parameters in the marine microalgal dry weight determination for laboratory-scale culture. The present findings could assist in developing a standardized protocol to ensure a high quality of biomass for microalgal studies. Microalgal biomass is one of the crucial criteria in microalgal studies. Many reported methods, even the well-established protocol on microalgal dry weight (DW) determination, vary greatly, and reliable comparative assessment amongst published results could be problematic. This study aimed to determine the best condition of critical parameters in marine microalgal DW determination for laboratory-scale culture using four different marine microalgal species. These parameters included the washing process, grades of glass microfiber filter (GMF), GMF pretreatment conditions, washing agent (ammonium formate) concentrations, culture: washing agent ratios (v:v) and washing cycles. GMF grade GF/A with precombustion at 450 °C provided the most satisfactory DW and the highest ash-free dry weight (AFDW)/DW ratio. Furthermore, 0.05 M ammonium formate with 1:2 culture: washing agent ratio and a minimum of two washing cycles appeared to be the best settings of microalgal DW determination. The present treatment increased the AFDW/DW ratio of the four respective microalgae by a minimum of 19%. The findings of this study could serve as a pivotal reference in developing a standardized protocol of marine microalgal DW determination to obtain veracious and reliable marine microalgal DW.

The Simultaneously Voltammetric Determination of Spinosad and Chlorantraniliprole Pesticides by Carbazole-Ferrocene Functionalized Carbon Nanotube Architecture

In this study, the carbon nanotube surface was modified by a novel compound bearing ferrocene as electroactive and carbazole as electropolymerizable groups by Click reaction for voltammetric determination of chlorantraniliprole and spinosad pesticides. An asymmetric ferrocene-carbazole compound (5) was synthesized and attached to the carbon nanotube surface (CNT) by the Click reaction for the preparation of the novel hybrid CNT material bearing ferrocene-carbazole groups (CFC). This hybrid material displayed great chemical stability and excellent sensitivity against chlorantraniliprole and spinosad pesticides. The linear ranges of chlorantraniliprole and spinosad were found 0.025–80.0 μM and 0.10–15.0 μM , respectively. The detection limits were 26 nM for chlorantraniliprole and 88 nM for spinosad. HPLC technique as a standard determination method was performed for accuracy of the voltammetric determination method in soil, apple and tomato samples. Compared with the chromatographic detection strategy, the method developed in this work is simple and easy to implement, which not only provides good selectivity and sensitivity for the detection of chlorantraniliprole and spinosad pesticides in real samples, but also opens up new perspectives for electrochemical sensing.

Multicomponent Internal Standard Methods for Determination of Vanadium, Chromium, Nickel, and Copper in Tool Steel Samples in Continuum-light-source Flame Atomic Absorption Spectrometry

This paper suggests an improved method for the sample preparation to quantify vanadium, chromium, nickel and copper in tool steel and high-speed steel by using flame atomic absorption spectrometry. The suggested method prepared two specimen solutions containing different internal standard elements: one included nickel for the quantifications of vanadium or copper and cobalt for the quantification of chromium and another included cobalt and phosphoric acid for the quantification of nickel, after the sample was digested with an acid mixture of hydrofluoric acid, nitric acid and phosphoric acid by using a microwave oven. A multi-wavelength high-resolution spectrometer system was employed to be simultaneously measured absorption lines for several pairs of an analyte and an internal standard element, such as vanadium and nickel, chromium and cobalt, nickel and iron, cobalt and phosphorus oxide, and copper and nickel. The internal standards contributed to more accurate and precise quantification of vanadium, chromium, nickel, and copper in tool steel and high-speed steel samples.

Ultrasensitive electrochemical molecularly imprinted sensor based on AuE/Ag-MOF@MC for determination of hemoglobin using response surface methodology.

Considering the importance of determining the levels of hemoglobin (Hb) as a vital protein in red blood cells, in this work a highly sensitive electrochemical sensor was developed based on a gold electrode (AuE) modified with Ag metal-organic framework mesoporous carbon (Ag-MOF@MC) and molecularly imprinted polymers (MIPs). To that end, the MIP layer was formed on the Ag-MOF@MC by implanting Hb as the pattern molecule during the polymerization. The modified electrode was designed using electrochemical approaches including differential pulse voltammetry (DPV), electrochemical impedance spectroscopy (EIS), and cyclic voltammetry (CV). Using a response level experimental design method, the most important parameters affecting the reaction of the sensing system including pH, incubation time, and scanning rate were optimized. Following the same route, the Hb concentration, pH, temperature, and elution times were optimized to prepare the imprinted polymer layer on the Ag-MOF@MC surface. By exploiting DPV techniques based on the optimal parameters, the electrochemical response of the AuE/Ag-MOF@MC-MIPs for Hb determination was recorded in a wide linear dynamic range (LDR) of 0.2 pM to 1000nM, with a limit of detection (LOD) of 0.09 pM. Moreover, the Ag-MOF@MC-MIP sensing system showed good stability, high selectivity, and acceptable reproducibility for Hb determination. The sensing system was successfully applied for Hb determination in real blood samples, and the results were compared with those of the standard methods for Hb determination. Acceptable recovery (99.0%) and RDS% (4.6%) confirmed the applicability and reliability of the designed Hb sensing system.

A portable analytical system for rapid on-site determination of total nitrogen in water

Excessive total nitrogen (TN) in the aqueous environment causes a notable negative impact on agriculture, human health, and the economy on a global scale. Conventional analytical techniques for determining TN in water usually involve long and tedious procedures with extensive sample preparation for digestion and titration. In recent years, lab-on-a-chip platforms have enabled in-situ measurements of water pollutants such as nitrate, nitrite, and ammonium. However, the digestion of organic nitrogen compounds in aqueous solutions still remains to be a challenge for portable analytical systems. In this paper, a portable TN analytical system is developed for on-site measurement of TN in a short assay time. It contains a TN reaction chamber for nitrogen digestion and reduction, and an optical measurement chamber for colorimetric determination of total nitrite. The ultraviolet-C (UVC)-thermal digestion method and the United States Environmental Protection Agency (EPA)-standard nitrate-nitrite determination method are implemented on the TN analytical system. Thorough investigations are performed to explore the optimum reaction conditions and reagent volumes in the process of TN oxidation, nitrate reduction, and nitrite detection, including oxidation time, temperature and substrate, oxidizer concentrations, nitrate reduction time, nitrite colorimetric reaction time, and reagents stability over time. Our system can complete fast oxidation and colorimetric determination of TN within 36 min, with a measurement range of 1 μg/L to 10 g/L and a limit of detection of 1.2 mg/L (lower than the World Health Organization standard of 10 mg/L). This portable TN analytical system enables the digestion and measurement of TN in a quick, portable, and low-cost manner.

Assessment of antinuclear antibodies (ANA): National recommendations on behalf of the Croatian society of medical biochemistry and laboratory medicine.

Antinuclear antibodies (ANA) represent a family of autoantibodies targeting ubiquitous cellular constituents and are a hallmark of systemic inflammatory autoimmune rheumatic diseases named connective tissue diseases (CTD). The gold standard method for ANA determination is indirect immunofluorescence (IIF) on the human laryngeal epidermoid carcinoma cell line type 2 substrate (HEp-2), but with increasing demand for ANA testing, novel methods eased for automation emerged, which allows testing by staff less experienced in this specific field of laboratory diagnostic. In 2016 The working group (WG) for laboratory diagnostics of autoimmune diseases as part of the Committee for the Scientific Professional Development of the Croatian Society of Medical Biochemistry and Laboratory Medicine (CSMBLM) published the data of a survey regarding general practice in laboratory diagnostics of autoimmune diseases in Croatia. Results indicated high diversity in the performance of autoantibody testing as well as reporting of the results and indicated the need of creating recommendations for the assessment of ANA that would help harmonize diagnostics of systemic autoimmune rheumatic diseases in Croatia. This document encompasses twenty-seven recommendations for ANA testing created concerning indications for ANA testing, preanalytical, analytical, and postanalytical issues, including rational algorithm and quality control assurance. These recommendations are based on the relevant international recommendations and guidelines for the assessment of ANA testing and relevant literature search and should help to harmonize the approach in ANA testing and clarify differences in interpretation of the results obtained using different methods of determination.

Evaluation of soil moisture by electrical resistivity in Oxisols of the central Brazilian savanna

The determination of the spatial distribution of soil moisture in the field is relevant for many purposes in agriculture and engineering, but it is still a challenge, since standard determination methods have limitations and problems of representativeness. Electrical resistivity may be used with this finality, but this method is still poorly studied for highly weathered tropical soils, which present structural and textural peculiarities. This work aims to present the calibration of the moisture/electrical resistivity relationships for a typical tropical soil, and to evaluate its robustness as a soil moisture prediction method. A highly weathered clay soil (Oxisol), representative of the Brazilian savanna (“Cerrado”), was sampled and the resistivity tests of the A, Bw and C horizons were conducted in a four-electrode cell array. The A and Bw horizons of clay Oxisols usually present resistivity values greater than temperate soils counterparts, which can be attributed to the lower content of highly active clays and higher macroporosity. The soil electrical resistivity/volumetric moisture relationship present a nonlinear and inverse trend and the generalized form of Archie's law has the potential to be applied to determine the water content of these soils. The electrical resistivity/volumetric soil moisture semi-log plot showed a two-step pattern, with a remarkable change in the behavior around the moisture associated to the wilting point. The field validation shows that the use of the electrical resistivity is possible for the measurement of soil moisture and for the subsurface mapping of its horizons.

Reappraisal of the ASTM/AASHTO Standard Rolling Device Method for Plastic Limit Determination of Fine-Grained Soils

Given its apparent limitations, various attempts have been made to develop alternative testing approaches to the standardized rolling-thread plastic limit (PLRT) method (for fine-grained soils), targeting higher degrees of repeatability and reproducibility. Among these, device-rolling techniques, including the method described in ASTM D4318/AASHTO T90 standards, based on original work by Bobrowski and Griekspoor (BG) and which follows the same basic principles as the standard thread-rolling (by hand) test, have been highly underrated by some researchers. To better understand the true potentials and/or limitations of the BG method for soil plasticity determination (i.e., PLBG), this paper presents a critical reappraisal of the PLRT–PLBG relationship using a comprehensive statistical analysis performed on a large and diverse database of 60 PLRT–PLBG test pairs. It is demonstrated that for a given fine-grained soil, the BG and RT methods produce essentially similar PL values. The 95% lower and upper (water content) statistical agreement limits between PLBG and PLRT were, respectively, obtained as −5.03% and +4.51%, and both deemed “statistically insignificant” when compared to the inductively-defined reference limit of ±8% (i.e., the highest possible difference in PLRT based on its repeatability, as reported in the literature). Furthermore, the likelihoods of PLBG underestimating and overestimating PLRT were 50% and 40%, respectively; debunking the notion presented by some researchers that the BG method generally tends to greatly underestimate PLRT. It is also shown that the degree of underestimation/overestimation does not systematically change with changes in basic soil properties; suggesting that the differences between PLBG and PLRT are most likely random in nature. Compared to PLRT, the likelihood of achieving consistent soil classifications employing PLBG (along with the liquid limit) was shown to be 98%, with the identified discrepancies being cases that plot relatively close to the A-Line. As such, PLBG can be used with confidence for soil classification purposes.

What’s in a wine? – A spot check of the integrity of European wine sold in China based on anthocyanin composition, stable isotope and glycerol impurity analysis

ABSTRACT The international wine market has been repeatedly hit by cases of fraud in recent decades. While several studies attested a special vulnerability of the fast growing wine business in China, reports on chemical analyses of commercial wine samples are rare. We examined 50 predominantly red wines with European labelling, which were purchased on the Chinese market, for fraud-relevant parameters. More than 20% of the tested samples revealed anomalies in relation to the stable isotope ratios of D/H, 18O/16O and 13C/12C, contents of technical glycerol by-products or anthocyanin composition. These results strongly suggested watering of the wines, chaptalisation, glycerol addition or the use of non-Vitis anthocyanin sources, respectively. Some of these samples also showed suspicious spelling errors or other irregularities in the labelling, but the majority appeared genuine to the eye. Hence, this spot check demonstrates the importance of chemical authenticity analysis of market samples in order to detect fraudulent products. Moreover, we used the same sample set for an evaluation of the Chinese standard method for carbon stable isotope determination of wine ethanol in comparison to the current OIV (International Organisation of Vine and Wine) standard method. The results of a Bland-Altman analysis indicated that the methods can be applied interchangeably. As the two methods differ in their workflow and in the requested equipment, this might eventually enable more laboratories to perform 13C/12C analysis of wine and spirits.

Determination of the scale mass on the surface of steel wire rod by metallographic method

A net detrimental effect of acid vapors on human health and environment characteristic for chemical methods of descaling the surface of steel wire rod, as well as the difficulties in the disposal of pickling sludge dictate the need for developing a new methodology free of the aforementioned shortcomings. A metallographic method for determining the mass of scale on the surface of a steel wire rod subjected to two-stage cooling is proposed. The tests were carried out in accordance with the requirements of current standards for assessing the accuracy (correctness and precision) of methods and measurement results. The proposed metallographic approach appeared advantageous for proved reliability of the scale mass determination with the required accuracy at a confidence level of 95%; for significant reduction in the material consumption due to the absence of the need for special sample preparation; reduction of the test time due to determination of metallographic parameters in parallel with optimization of the descaling procedure through scale dissolution in a pickling solution and protection of the base metal with the inhibitors; for elimination of using acids and, as a result, the necessity of waste disposal. Apart from quantifying the scale content, the method provides for determination of the phase component ratio. It is shown that the metallographic method, including determination of the scale mass by measuring the thickness of the layers of the scale phases (wustite, magnetite), can be used as an alternative to the traditional standardized method for rapid determination of the scale amount and X-ray structural analysis of the phase composition.

Quench me if you can: Alpha-2-macroglobulin trypsin complexes enable serum biomarker analysis by MALDI mass spectrometry

One of the main functions of alpha-2-macroglobulin (A2M) in human blood serum is the binding of all classes of protease. It is known that trypsin, after such interaction, possesses modified proteolytic activity. Trypsin first hydrolyzes two bonds in A2M’s ‘bait region’, and the peptide 705VGFYESDVMGR715 is released from A2M. In this work, specifics of the A2M-trypsin interaction were used to determine A2M concentration directly in human blood serum using MALDI mass-spectrometry. Following exogenous addition of trypsin to human blood serum in vitro, the concentration of the VGFYESDVMGR peptide was measured, using its isotopically-labeled analogue (18O), and A2M concentration was calculated. The optimized mass spectrometric approach was verified using a standard method for A2M concentration determination (ELISA) and the relevant statistical analysis methods. It was also shown that trypsin’s modified proteolytic activity in the presence of serum A2M can be used to analyze other serum proteins, including potential biomarkers of pathological processes. Thus, this work describes a promising approach to serum biomarker analysis that can be technically extended in several useful directions.

Strain depth profiles in thin films extracted from in-plane X-ray diffraction

Thin films generally contain depth-dependent residual stress gradients, which influence their functional properties and stability in harsh environments. An understanding of these stress gradients and their influence is crucial for many applications. Standard methods for thin-film stress determination only provide average strain values, thus disregarding possible variation in strain/stress across the film thickness. This work introduces a new method to derive depth-dependent strain profiles in thin films with thicknesses in the submicrometre range by laboratory-based in-plane grazing X-ray diffraction, as applied to magnetron-sputtering-grown polycrystalline Cu thin films with different thicknesses. By performing in-plane grazing diffraction analysis at different incidence angles, the in-plane lattice constant depth profile of the thin film can be resolved through a dedicated robust data processing procedure. Owing to the underlying intrinsic difficulties related to the inverse Laplace transform of discrete experimental data sets, four complementary procedures are presented to reliably extract the strain depth profile of the films from the diffraction data. Surprisingly, the strain depth profile is not monotonic and possesses a complex shape: highly compressive close to the substrate interface, more tensile within the film and relaxed close to the film surface. The same strain profile is obtained by the four different data evaluation methods, confirming the validity of the derived depth-dependent strain profiles as a function of the film thickness. Comparison of the obtained results with the average in-plane stresses independently derived by the standard stress analysis method in the out-of-plane diffraction geometry validates the solidity of the proposed method.

Determination of lipophilic marine biotoxins by liquid chromatography-tandem mass spectrometry in five shellfish species from Washington State, USA

Low extraction efficiency (60-81%) of okadaic acid (OA) and dinophysistoxin 1 (DTX1) was obtained for 4 out of 5 shellfish species from Washington State (WA), USA, during application of a standard extraction method for determination of lipophilic marine biotoxins by LC-MS/MS as recommended by the European Union Reference Laboratory for Marine Biotoxins (EURLMB). OA and total OA including esters, DTX1, DTX2, and total DTX including esters, azaspiracid 1, 2, and 3 (AZA1, AZA2, and AZA3), pectenotoxin 2 (PTX2), and yessotoxin (YTX) were the toxins examined. Matrix-matched standards prepared from the same control samples used for spike-and-recovery tests were employed to evaluate toxin extraction efficiency and sample clean-up procedures. We adjusted the EURLMB extraction method by either using an acidified methanol extraction or pre-cooking shellfish homogenates at 70 °C for 20 min before EURLMB extraction. Extraction efficiency was improved markedly for OA and DTX1 with both modified methods and for YTX with the pre-cooking step included. However, recoveries were lower for YTX using the acidified methanol extraction and for PTX2 in non-mussel samples with the pre-cooking step. A hexane wash was applied to clean water-diluted non-hydrolyzed samples and a hexane wash was combined with solid-phase extraction for cleaning hydrolyzed samples. Improved sample clean-up, combined with LC-MS/MS adjustments, enabled quantification of U.S. Food and Drug Administration-regulated toxins in five shellfish species from WA with acceptable accuracy using non-matrix matched calibration standards.

Nutlets of Tilia cordata Mill. and Tilia platyphyllos Scop. – Source of bioactive compounds

The aim of this study was to analyse contents of phytosterols, carotenoids, tocochromanols and fatty acid composition in oil from nutlets of linden. Standard determination methods for these compounds were applied using liquid and gas chromatography. In the analysed oils from small and large-leaved lime the dominant acids included linoleic (53.1 and 54.1%), oleic (18.5 and 22.2%), palmitic (15.3 and 11.8%). Linden seed oil was characterised by very high contents of phytosterols (2–2.5 g/100 g). β-Sitosterol was the dominant phytosterol (80–83%). Additionally squalene was detected at 806 and 607 mg/100 g, respectively, for Tilia cordata and T.platyphyllos. It was found that linden seed oil contains tocopherols (93%) and tocotrienols (7%). In terms of tocochromanol contents this oil is surpassed only by wheat germ oil. In terms of the α-T equivalent these oils contain 95 (Tilia cordata) and 50.6 mg/100 g vitamin E (Tilia platyphyllos)