A comparison between the enzymatic oxidation method and headspace gas chromatography with a flame ionization detector in the determination of postmortem blood ethanol.

Ethanol is the most commonly encountered substance in forensic toxicology. Determining blood alcohol concentration (BAC) in autopsies accounts for the majority of work in forensic diagnosis. The most common method to assess BAC is the enzymatic oxidation method because of its low cost, easy operation, and high throughput. Still, the elevated lactate and lactate dehydrogenase (LDH) levels in postmortem blood may affect accuracy. This study uses headspace gas chromatography with a flame ionization detector (HS-GC/FID) to assess the interference of lactate and LDH levels on BAC in 110 autopsied blood samples determined by the enzymatic oxidation method. The results showed that lactate and LDH levels in postmortem blood were higher than in normal blood. There was a weak correlation between the lactate levels and BAC difference (r = 0.23, p < 0.05) and a strong correlation between LDH levels and BAC difference (r = 0.67, p < 0.001). The differentiation of BAC between the enzymatic oxidation method and HS-GC/FID was significant (p < 0.001), confirming the interference significantly. All postmortem blood samples with lactate and LDH levels higher than regular lead to a positive error in determining BAC by enzymatic oxidation method. The study results suggest that the HS-GC/FID method should be used to determine BAC in postmortem blood samples instead of the enzymatic oxidation method to avoid mistakes in forensic diagnosis.

A high-performance method for quantitation of aflatoxins B1, B2, G1, G2: Full validation for raisin, peanut matrices, and survey of related products at Ho Chi Minh City.

Optimization and validation for simultaneous quantitation of four aflatoxins B1, B2, G1, and G2 in peanuts and raisins were performed on ultra-performance liquid chromatography in a combination of fluorescence detector, without derivatization. The advantages were short analysis time, simple sample handling, and reduced solvent consumption. Instrument detection limits of AFB1, AFB2, AFG1, and AFG2 were 0.07, 0.01, 0.1, and 0.008 µg/kg, respectively, lower than those obtained by LCMSMS and HPLC-FLD with derivatization. Two solvent mixtures were chosen for two different matrices whose matrix effect was not negligible (2.81%-8.04% for peanuts and 5.63%-11.43% for raisins). The linear ranges were from 0.2 to 20 µg/L for AFB1 and AFG1 and from 0.05 to 5 µg/L for AFB2 and AFG2. The limits of detection and quantification were 0.025-0.1 and 0.075-0.3 µg/kg for peanuts and raisins, respectively. Recoveries at three other concentrations from 0.75 to 125 µg/kg of total aflatoxins were obtained between 76.5% and 99.8% (with RSD < 6%) following the SANTE 11312/2021. Validation parameters complied with the requirements of ISO/IEC 17025:2017. The extracts and the sample could be stabilized at 4°C and 20°C for 24 h and at -20°C for up to 21 days, respectively. Thus, the study can be used as a standard method for the analysis of Aflatoxins (AFs) in peanut and raisin matrices. Investigation of 350 peanut samples collected at Markets in the central districts of HCM city showed that 28.6% were contaminated with AFB1 from 0.31 up to 554 µg/kg; 13.4% contained AFB2, and 5.7% of AFG1 in the range of 0.4-53 µg/kg and 0.4-9.57 µg/kg, respectively; AFG2 (about 0.6%) was detected from 0.45 to 0.75 µg/kg. Meanwhile, 12.8% exceeded the total aflatoxins limit, and 13.4% exceeded the AFB1 limit. AFs were almost not found in the 350 raisin samples.

Investigation of arsenic contamination in groundwater using hydride generation atomic absorption spectrometry.

The validated hydride generation atomic absorption spectrometry (HG-AAS) method has been used to investigate total arsenic in groundwater. Under optimal experimental conditions, the concentration of arsenic in groundwater can be analysed in the range of 0.5 to 50 µg/L, with a method detection limit of 0.15 µg/L. Its recovery in the field is from 96.3 to 99.8%, with high repeatability. The method was used to observe the total arsenic pollution in groundwater collected in Phu Tho Province, Vietnam. A total of 364 groundwater samples were analysed. The results showed that arsenic pollution was significant, with 15.93% of the samples higher than the maximum permissible level of arsenic. About 20.69% of the contaminated samples had a total arsenic ten times higher (100 µg/L) than the maximum permissible level of arsenic. The pollution source was also considered by comparing the arsenic level in the groundwater with arsenic in the surface water in the same areas. Thus, the use of the high-accuracy and sensitive method, HG-AAS, supplies valuable data on groundwater pollution for water resources management and environmental protection.

Comparison of Microplastic Pollution in Beach Sediment and Seawater at UNESCO Can Gio Mangrove Biosphere Reserve.

Microplastics have become a global concern due to their persistent properties and impacts on the marine environment. This research investigates pollution sources and behaviors of microplastics at UNESCO Can Gio Mangrove Biosphere Reserve. Density flotation with sodium chloride is employed to extract microplastics from sand at Can Gio Beach, and a double-filtration procedure is developed to recover microplastics from seawater at the beach and Dong Tranh Cape. The microplastics' morphology and type are analyzed by micro-Raman spectroscopy. The results show that microplastics are accumulated at concentrations from 31.99 to 92.56 MPs g-1 at various sand layers. The seawater at Can Gio Beach and Dong Tranh Cape contains 6.44 and 3.75 MPs L-1 of microplastics, respectively. White polyethylene fragments predominate, and all the microplastics comprise small secondary microplastics with a minimum size of 25 µm and a maximum size of 260 µm for fragments and a length of 640 µm for fibers. The proportions of polyethylene, polypropylene, polystyrene, and polymethylmethacrylate are similar. The differing percentages of other compositions in sand and seawater are attributed to the morphology and density of the microplastics. The results indicate the extent of microplastic pollution and suggest appropriate strategies for tourism development at the Biosphere Reserve.

Fluorescence Determination of Peptidase Activity Based on the Quenching of a Fluorophore-Labelled Peptide by Graphene Oxide.

In this study, a fluorescence detection strategy is reported for the peptidase activity assay, which is based on fluorescence resonance energy transfer (FRET) from a fluorophore-labelled peptide to graphene oxide (GO). By the hydrolysis of the peptide, the fluorophore-labelled peptide releases the fluorophore 5-carboxyfluorescein, which can avoid quenching from GO. Thus, the increased intensity of the obtained fluorescence signal in the assay is directly dependent on the peptidase activity. As a model case of the developed strategy, the activity determination of pancreatic elastase (PE) is performed. Under the optimal experimental conditions at an excitation wavelength of 494 nm, the activity of PE can be determined in the range from 0.003 to 0.10 U/mL, with a detection limit of 0.001 U/mL at the emission wavelength of 518 nm. This is ultra-sensitive for the determination of PE. The specificity of the method is demonstrated by the analysis of PE under complex conditions using fetal bovine serum as the substrate. Hence, the developed method might provide an intrinsically convenient, sensitive platform for the PE activity assay and related biochemical studies due to its homogeneous, and fluorescence-based detection strategy.

A Validated Method for the Simultaneous Determination of Methamphetamine and 3,4-Methylenedioxy-N-methamphetamine in Blood-Based Liquid Chromatography-Tandem Mass Spectrometry.

A liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) method has been validated for the simultaneous determination of methamphetamine (MA) and 3,4-methylenedioxy-N-methamphetamine (MDMA) in the blood sample. Under the optimal experimental conditions, the concentration of MA can be determined in the range from 1 µg/L to 5000 µg/L with the method detection limit (MDL) of 0.31 µg/L. The range from 0.5 to 500 µg/L is observed for the determination of MDMA with the MDL down to 0.25 µg/L. The practical applicability of the method is performed with the recovery ranging from 85.3% to 94% for MA and from 86.9% to 95.5% for MDMA. At the different concentrations of drugs, the relative standard deviations (RSD) for both MA and MDMA are lower than 5.7%. The method was applied to analyse 1995 blood samples that had been collected from the Forensic Medicine Centre of Ho Chi Minh City. The results showed 1.75% positive with MA and 0.25% positive with MDMA. These two drugs take 10% of the total drugs positive samples. By using deuterium-labelled methamphetamine-d5 and 3,4-methylenedioxy-N-methamphetamine-d5 as the internal standards in the determination and the use of MS/MS in multiple reaction monitoring mode signal readout, the method exhibits robustness specificity and can be applied in simultaneous determination of MA and MDMA in blood with high selectivity and sensitivity.

Fluorescence determination of the activity of O6-methylguanine-DNA methyltransferase based on the activation of restriction endonuclease and the use of graphene oxide.

A fluorescence method is described for the determination of the activity of O6-methylguanine-DNA methyltransferase (MGMT). It is based on the activation of restriction endonuclease PvuII and the adsorbing a fluorophore-labelled DNA onto the surface of graphene oxide (GO). MGMT activity removes the methyl group from O6-methylguanine (O6MeG) in the fluorophore-labelled DNA to unblock the specific recognition site for further hydrolysis reaction of restriction endonuclease PvuII. The endonuclease catalytic reaction releases fluorophores (5-carboxyfluorescein) from fluorophore-labelled DNA, which can avoid fluorescence quenching by GO, creating an abundance of the fluorescence signal. The fluorescence increase in the assay is thus directly dependent on the MGMT activity. Under the optimal conditions with the emission wavelength of 519 nm (exitation at 494 nm), the activity of the MGMT can be determined in the range 0.5 to 35 ng mL-1 with a detection limit of 0.15 ng mL-1. This is extremely sensitive for the determination of MGMT. The short time of analysis (2 h) is superior to many reported strategies. The method can also be extended for the rapid and sensitive activity assay of other DNA repair enzymes by designing a proper substrate DNA. Conceivably, the technique represents a powerful tool for diagnosis and drug exploitation. Graphical abstract Schematic representation of the fluorescence method for MGMT activity assay.

Immunoreaction-Mediated Aggregation of Gold Nanoparticles for Sensitive and Selective Assay of Hepatitis B Surface Antigen.

A rapid, sensitive and quantitative assay method for Hepatitis B surface antigen (HBsAg) is of paramount importance for the drug development and in the diagnosis of this disease. Here, we proposed a novel biosensor that sensitively and selectively screen Hepatitis B surface antigen. This strategy relies on the cross-linking aggregation of gold nanoparticles (AuNPs) that were decorated with Hepatitis B surface antibody (HBsAb) and Raman reporter 5-thio-nitrobenzoic acid (TNB) by Hepatitis B surface antigen. The immune reaction between HBsAb and HBsAg offers this strategy high specificity, and the use of AuNPs additionally allows a visual and homogeneous assay format, thus permitting improved simplicity and throughput of the assays. The selectivity and sensitivity in HBsAg assay were achieved with a wide linear response range from 0.5 ng/mL to 50 ng/mL and a detection limit of 0.2 ng/mL. The results indicated that this strategy can offer a simple, robust and convenient platform for HBsAg analysis and related biochemical studies with high sensitivity and selectivity.

Target-mediated consecutive endonuclease reactions for specific and sensitive homogeneous fluorescence assay of O6-methylguanine-DNA methyltransferase.

O(6)-Methylguanine-DNA methyltransferase (MGMT) is one of the most important DNA-repair enzymes. Herein, a simple, sensitive and selective homogeneous fluorescence assay strategy is developed for the detection of MGMT on the basis of target-mediated two consecutive endonuclease reactions. The activity assay of MGMT is firstly accomplished using a hairpin-structured DNA substrate to offer a specific recognition site on the substrate DNA for restriction endonuclease PvuII, and thus to initiate the first endonuclease reaction. The product which activates the second endonuclease reaction allows an efficient amplification approach to create an abundance of fluorescence signal reporters. The first endonuclease reaction offers the method high specificity and the second one furnishes the assay improved sensitivity. The results reveal that the MGMT assay strategy shows dynamic responses in the concentration range from 1 to 120 ng mL(-1) with a detection limit of 0.5 ng mL(-1). By simply altering the alkylated bases, this strategy can also be extended for the detection of other alkyltransferases. Therefore, the developed strategy might provide an intrinsically convenient, sensitive and specific platform for alkyltransferase activate assay and related biochemical studies due to its label-free, homogeneous, and fluorescence-based detection format.

Proteolysis-mediated protection of gold nanoparticles for sensitive activity assay of peptidases.

Rapid, sensitive and quantitative assays for peptide hydrolysis enzymes are of paramount importance for drug development and in the diagnosis of disease. Here, we proposed a novel biosensor for sensitive and selective active screening of peptidases. This strategy relies on the proteolysis-mediated protection of gold nanoparticles (AuNPs) that were decorated with biotin-labeled substrate peptides and can be aggregated by streptavidin. Enzyme-mediated protection of AuNPs offers this strategy high specificity, and the use of AuNPs additionally allows a visual and homogeneous assay format, thus permitting improved simplicity and throughput of the assays. As a model case, desirable selectivity and sensitivity in peptidase assay were achieved in the active assays of pancreatic elastase with a wide linear response range from 0.005 to 0.10 U/mL and a detection limit of 0.003 U/mL. The results indicated that this strategy can offer a simple, robust and convenient platform for visualized peptidase activity analysis and related biochemical studies with high sensitivity and selectivity.

Enzyme-catalyzed assembly of gold nanoparticles for visualized screening of DNA base excision repair.

Activity screening of DNA base excision repair (BER) enzymes is a crucial step for understanding numerous fundamental biochemical processes. A novel label-free homogeneous technique is developed for visualized uracil-DNA glycosylase (UDG) activity assay using gold nanoparticles (AuNPs). This strategy relies on the enzyme-catalyzed assembly of AuNPs decorated with DNA probes. In the presence of endonuclease IV (an enzyme which can further hydrolyze the products from UDG-catalyzed reaction), the substrate DNA selectively interacts with UDG followed by the efficient release of a single-strand probe. The released single-strand probe then makes the network-like assembly of decorated AuNPs to provide a visible signal for UDG activity. This strategy that can be performed in a label-free homogeneous assay format improved the duration, the simplicity and the throughput of UDG activity screening. The results provided in the present study revealed that this strategy could hold great potential as a robust, convenient and visualized platform for activity screening of uracil-DNA glycosylases with high selectivity and desirable sensitivity.