The focus of this work was the characterisation of an impurity sometimes found in batches of the hydroxyxanthene colour additives D&C Red No. 27 (R27) and D&C Red No. 28 (R28, phloxine B). Countercurrent chromatography (CCC) was used to separate and concentrate the impurity (named here pHXCA) to similar to 48% from a dye sample that contained similar to 0.74% pHXCA. Pure pHXCA was obtained by collecting analytical HPLC-separated fractions from multiple injections of the CCC-concentrated sample. Various NMR and mass spectral techniques were used to determine the structure of pHXCA. In particular, the presence of a 35/37Cl isotope shift in the 13C NMR spectrum pointed to the location of the C-Cl bond specific to pHXCA and facilitated identification of its structure as 1-carboxy-7-bromo-6-hydroxy-2,3,4,5-tetrachloroxanthone. Significantly, that structure was different from the one erroneously reported by other researchers. A rapid screening of 18 dye batches (R27, n = 13; R28, n = 5) representing nine manufacturers, revealed that 12 batches contained pHXCA, with levels that ranged from 0.0009 to 0.182%. A side reaction during the manufacture of R27/28 in which a bromine atom is replaced with a chlorine atom in a closely related contaminant is suggested as the potential basis of pHXCA formation.
Perchlorate and chlorate are environmental contaminants that competitively inhibit thyroidal iodide uptake, posing health risks particularly for vulnerable populations. This study developed an isotope dilution UPLC-MS/MS method to determine perchlorate and chlorate levels in 346 samples (vegetables, fruits, eggs, and drinking water) collected from Shenzhen. Probabilistic dietary exposure assessment was conducted across 11 age-gender groups using Monte Carlo simulation (10,000 iterations) with local consumption data (n = 853). Perchlorate was detected in 94% of samples (mean = 16.1 mu g/kg), with leafy vegetables (78.5 mu g/kg) and eggs (32.0 mu g/kg) showing the highest mean levels; chlorate was detected in 45% of samples, whilst drinking water had a 100% detection rate and the highest concentrations (mean = 53.5 mu g/L). Males aged 4-14 years had the highest 95th percentile perchlorate estimated daily intake (EDI) (1.19 mu g/kg bw/day), with 4.0% exceeding the EFSA tolerable daily intake (TDI). For chlorate, the highest 95th percentile EDI was observed in the 0-3 years old group (14.8 mu g/kg bw/day), with 55.2% exceeding the TDI under the lower bound scenario. Sensitivity analysis identified perchlorate concentration in leafy vegetables and drinking water intake as the key drivers of perchlorate and chlorate exposure, respectively. These findings indicate widespread contamination and notable health risks for children, supporting the need for regulatory measures to reduce chlorate in drinking water and monitor perchlorate in leafy vegetables and eggs.
In recent years, the United States Food and Drug Administration (US FDA) has documented several instances where more abundant, and lower priced, jumbo squid (Dosidicus gigas) has been substituted for octopus (family Octopodidae) in a variety of products (e.g. frozen, canned) resulting in the products being misbranded and/or adulterated. FDA typically utilises a short (approx. 655 base pair) fragment of the cytochrome c oxidase subunit 1 (COI) mitochondrial gene (a.k.a. DNA barcoding) for the identification of seafood products. However, this method is not applicable for the identification of canned products due to DNA fragmentation that occurs during the retort process. Described herein is a modified DNA barcoding method using standard DNA marine invertebrate barcoding primers in combination with degenerate forward and reverse internal primers to create two smaller (313-316 base pair) fragments. The fragments are sequenced separately then assembled to form a nearly full-length DNA barcode fragment. Using this method, 11 of the 12 canned jumbo squid and octopus products tested, in a variety of forms (i.e. in hot sauce, in garlic sauce, smoked, etc.), were successfully identified. This method is fit for the purpose of ad hoc regulatory identification of canned squid and octopus products to confirm product labelling or to be used as a screening tool to prevent species substitution, economic adulteration, and fraud.
Mercury toxicity depends strongly on chemical form, yet dietary exposure assessments and regulatory monitoring are typically based on total mercury (THg). This study evaluated inorganic mercury (iHg), methylmercury (MeHg), THg, selenium (Se), and Hg- and Se-containing nanoparticles (NPs) in 44 seafood and 10 non-seafood products from the Danish market. A rapid reversed-phase HPLC-ICP-MS method was validated in-house and applied for Hg speciation, providing baseline separation of iHg and MeHg within 3.5 min. MeHg recoveries in certified reference materials were 94-101%, within-laboratory reproducibility was <= 12.3%, and the limit of quantification (LOQ) for fresh seafood samples was 0.29 & micro;g kg-1 wet weight. The sum of speciated Hg (& sum;Hg = iHg + MeHg) agreed with acid-digestion THg within 80-105% across seafood samples. In fish and crustaceans, MeHg was the predominant Hg species, accounting for approximately 90-94% of THg, supporting the use of THg as a practical proxy for MeHg exposure in these categories. In contrast, MeHg accounted for only 22.6-44.5% of THg in mussels and was below the LOQ in all non-seafood matrices except one pig liver sample, demonstrating that fixed species-proportion assumptions may overestimate MeHg exposure in some food categories. The highest THg concentration was observed in common dab (Limanda limanda) at 0.33 mg kg-1 wet weight, slightly above the 0.30 mg kg-1 EU maximum level applicable to this species group under Commission Regulation (EU) 2023/915. Hg-containing NPs represented approximately 1% of the iHg fraction within the detectable particle size range across analysed samples, indicating a minor contribution to the measured iHg pool.
Short-chain fructooligosaccharides (scFOS) are prebiotic food ingredients, and 1-kestose-enriched preparations are of interest because of their defined short-chain composition. This study evaluated a beta-fructofuranosidase preparation from Aspergillus fijiensis SYG-K1 developed for 1-kestose-enriched scFOS production. Fermented food-derived microbial isolates were screened for FOS-producing activity, and the selected parental strain was improved by ultraviolet/N-methyl-N '-nitro-N-nitrosoguanidine-induced random mutagenesis. The resulting SYG-K1 strain showed increased beta-fructofuranosidase activity and produced a favourable FOS profile with high GF2 formation, limited GF3 formation, and no detectable GF4 formation under the tested conditions. A GLP-compliant toxicological safety package was applied according to appropriate OECD test guidelines, including acute oral toxicity, repeated-dose oral toxicity, and genotoxicity studies. A single oral dose of 2,000 mg/kg produced no toxicity. Repeated oral dose administration of up to 1,643.9 mg total organic solids (TOS)/kg bw/day produced no treatment-related adverse effects or delayed toxicity. The enzyme preparation was negative in bacterial reverse mutation, in vitro chromosomal aberration, in vitro micronucleus, and in vivo micronucleus assays. The NOAEL was established at 1,643.9 mg TOS/kg bw/day, supporting the toxicological safety of this enzyme preparation.
