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Supplementing fish oil is one of the strategies to reduce the risk of cardiovascular disease, the leading cause of death around the world. Contradictorily, fish oil may also contain trimethylamine-N-oxide, a recently emerged risk factor for cardiovascular disease, as well as one of its precursors, trimethylamine. A method suitable for routine quantification of trimethylamine-N-oxide and trimethylamine in fish oil with a quick and easy liquid extraction without derivatization has been developed. Liquid chromatography with tandem mass spectrometry detection was employed along with a zwitterionic hydrophilic interaction liquid chromatography column and a gradient elution with eluents containing 50 mmol/L of ammonium formate. An internal standard (triethylamine) was used for quantification by mass spectrometry with an external calibration. The assay proved high linearity in the ranges of 10 to 100 ng/mL and 100 to 1000 ng/mL for trimethylamine-N-oxide and trimethylamine, respectively. The lowest limit of quantification was determined to be 100 µg/kg for trimethylamine and 10 µg/kg for trimethylamine-N-oxide, with the limit of detection at 5 µg/kg and 0.25 µg/kg, respectively. Accuracy ranged from 106–119%. Precision was below 7% the relative standard deviation for both analytes. The method was successfully applied for the determination of trimethylamine-N-oxide and trimethylamine contents in nine commercially available liquid fish oils and three commercially available fish oil capsules, showing that trimethylamine and trimethylamine-N-oxide are not present in highly refined fish oils. [ABSTRACT FROM AUTHOR]

Ningnanmycin is a widely used antibiotic in agricultural production that effectively controls fungal and viral diseases in tea trees and chrysanthemums. The polarity characteristic of ningnanmycin has posed limitations on the development of robust detection methods, thereby hindering effective monitoring and control measures. By combining cation exchange solid phase extraction (SPE) with hydrophilic interaction chromatography tandem mass spectrometry (HILIC-MS/MS), we have effectively tackled the issue pertaining to the separation and retention of ningnanmycin. The average recoveries of ningnanmycin in green tea, black tea, and chrysanthemum were 77.3–82.0%, 80.1–81.5%, and 74.0–80.0%, respectively. The intraday and interday relative standard deviations (RSDs) were below and equal to 7.7%. Good linearity was observed in the concentration range of 1–1000 μg/L (R2 > 0.998). The limits of detection (LODs) ranged from 1.1 μg/kg to 7.1 μg/kg, and the limits of quantification (LOQs) ranged from 3.6 μg/kg to 23.7 μg/kg for ningnanmycin. These results indicate the good accuracy, repeatability, reproducibility, and sensitivity of the method. It is suitable for detecting ningnanmycin in tea and chrysanthemum. [ABSTRACT FROM AUTHOR]

Carob (Ceratonia siliqua) is an exceptional source of significant bioactive compounds with great economic importance in the Mediterranean region, where it is widely cultivated. Carob fruit is used for the production of a variety of products and commodities such as powder, syrup, coffee, flour, cakes, and beverages. There is growing evidence of the beneficial effects of carob and the products made from it on a range of health problems. Therefore, metabolomics could be used to explore the nutrient-rich compounds of carob. Sample preparation is a crucial step in metabolomics-based analysis and has a great impact on the quality of the data obtained. Herein, sample preparation of carob syrup and powder was optimized, to enable highly efficient metabolomics-based HILIC-MS/MS analysis. Pooled powder and syrup samples were extracted under different conditions by adjusting pH, solvent type, and sample weight to solvent volume ratio (Wc/Vs). The metabolomics profiles obtained were evaluated using the established criteria of total area and number of maxima. It was observed that the Wc/Vs ratio of 1:2 resulted in the highest number of metabolites, regardless of solvent type or pH. Aqueous acetonitrile with a Wc/Vs ratio of 1:2 satisfied all established criteria for both carob syrup and powder samples. However, when the pH was adjusted, basic aqueous propanol 1:2 Wc/Vs and acidic aqueous acetonitrile 1:2 Wc/Vs provided the best results for syrup and powder, respectively. We strongly believe that the current study could support the standardization of the metabolomics sample preparation process to enable more efficient LC-MS/MS carob analysis. [ABSTRACT FROM AUTHOR]

In recent years, the consumption of blue-green algae (BGA) dietary supplements is increasing because of their health benefits. However, cyanobacteria can produce cyanotoxins, which present serious health risks. In this work we propose hydrophilic interaction liquid chromatography coupled with tandem mass spectrometry (HILIC-MS/MS) to determine cyanotoxins in BGA dietary supplements. Target toxins, including microcystin-leucine-arginine (MC-LR) and microcystin-arginine-arginine (MC-RR), nodularin, anatoxin-a and three non-protein amino acids, β-N-methylamino-L-alanine (BMAA), 2,4-diaminobutyric acid (DAB) and N-(2-aminoethyl)glycine (AEG), were separated using a SeQuant ZIC-HILIC column. Cyanotoxin extraction was based on solid–liquid extraction (SLE) followed by a tandem-solid phase extraction (SPE) procedure using Strata-X and mixed-mode cation-exchange (MCX) cartridges. The method was validated for BGA dietary supplements obtaining quantification limits from 60 to 300 µg·kg−1. Nine different commercial supplements were analyzed, and DAB, AEG, and MCs were found in some samples, highlighting the relevance of monitoring these substances as precaution measures for the safe consumption of these products. [ABSTRACT FROM AUTHOR]

RNA methylation plays a vital role in the pathogenesis of a variety of diseases including cancer, and aberrant levels of modified nucleosides in RNA were revealed to be related to cancer. Urine is a favored source for biomarker discovery due to the non-invasion to patients. Herein, we developed a sensitive hydrophilic interaction liquid chromatography tandem mass spectrometry (HILIC–MS/MS) method combined with stable isotope dilution for accurate quantification of methylated nucleosides in human urine. With this method, we successfully quantified ten methylated nucleosides in urine samples collected from healthy controls and breast cancer patients. We found N6-methyladenosine (m6A), 2′-O-methyladenosine (Am), N1-methyladenosine (m1A), N6,2′-O-dimethyladenosine (m6Am), N1-methylguanosine (m1G), 2′-O-methylguanosine (Gm), 5-methylcytidine (m5C) and 2′-O-methylcytidine (Cm) were all decreased in early-stage breast cancer patients, and a nomogram prediction model was constructed. Locally advanced breast cancer patients exhibited elevated levels of urinary 2′-O-methylated nucleosides in comparison to early-stage breast cancer patients. Together, we developed a robust method for the simultaneous determination of methylated nucleosides in human urine, and the results revealed an association between the contents of urinary methylated nucleosides and the occurrence of breast cancer, which may stimulate future studies about the regulatory roles of these methylated nucleosides in the initiation and progression of breast cancer. [ABSTRACT FROM AUTHOR]

Microbiome-derived trimethylamine N-oxide (TMAO) has been associated with platelet hyperreactivity and subsequent atherogenesis. Whether physiological TMAO-levels influence platelet-derived lipid mediators remains unknown. Little is known about pre-analytic factors potentially influencing TMAO concentrations. We aimed at developing a quantitative LC-MS/MS method to investigate in-vivo and in-vitro pre-analytical factors in TMAO analysis to properly assess the proposed activating effect of TMAO on platelets. TMAO, betaine, carnitine, and choline were analyzed by HILIC-ESI-MS/MS within 6 min total run time. Method validation included investigation of reproducibility, recovery, sensitivity, and in-vitro pre-analytical factors. A 24-h monitoring experiment was performed, evaluating in-vivo pre-analytical factors like daytime or diet. Finally, the effects of different TMAO concentrations on platelet activation and corresponding alterations of platelet-derived eicosanoid release were analyzed. The method showed high reproducibility (CVs ≤ 5.3%), good recovery rates (96–98%), and negligible in-vitro pre-analytical effects. The influence of in-vivo pre-analytical factors on TMAO levels was not observable within the applied experimental conditions. We did not find any correlation between TMAO levels and platelet activation at physiological TMAO concentrations, whereas platelet-derived eicosanoids presented activation of the cyclooxygenase and lipoxygenase pathways. In contrast to previously published results, we did not find any indications regarding diet dependency or circadian rhythmicity of TMAO levels. Our results do not support the hypothesis that TMAO increases platelet responsiveness via the release of lipid-mediators. [ABSTRACT FROM AUTHOR]

Adenosine is a purine ribonucleoside with important roles in various physiological processes. A number of studies have indicated the importance of adenosine in cardiovascular diseases including syncope; however, the accurate determination of adenosine in human blood is challenging due to the molecule's instability. In the present study, we report a simple method for the pre-treatment of blood samples and the development of a fast and efficient hydrophilic interaction chromatographic tandem mass spectrometry method for the analysis of adenosine in patient blood. During collection, samples were mixed directly with a solvent mixture containing 95% acetonitrile and 10 mM ammonium formate in a Vacutainer tube, resulting in successful prevention of adenosine metabolic processes and direct blood sample deproteinization. The method was validated according to bioanalytical industry guidelines and found to be accurate, repeatable, specific and sensitive with LLOQ 0.005 μg/mL, thus allowing its application in the analysis of real clinical samples. [ABSTRACT FROM AUTHOR]

Tetrodotoxins (TTXs) are traditionally associated with the occurrence of tropical Pufferfish Poisoning. In recent years, however, TTXs have been identified in European bivalve mollusc shellfish, resulting in the need to assess prevalence and risk to shellfish consumers. Following the previous identification of TTXs in shellfish from southern England, this study was designed to assess the wider prevalence of TTXs in shellfish from around the coast of the UK. Samples were collected between 2014 and 2016 and subjected to analysis using HILIC-MS/MS. Results showed the continued presence of toxins in shellfish harvested along the coast of southern England, with the maximum concentration of total TTXs reaching 253 µg/kg. TTX accumulation was detected in Pacific oysters (Crassostrea gigas), native oysters (Ostrea edulis) common mussels (Mytilus edulis) and hard clams (Mercenaria mercenaria), but not found in cockles (Cerastoderma edule), razors (Ensis species) or scallops (Pecten maximus). Whilst the highest concentrations were quantified in samples harvested during the warmer summer months, TTXs were still evident during the winter. An assessment of the potential causative factors did not reveal any links with the phytoplankton species Prorocentrum cordatum, instead highlighting a greater level of risk in areas of shallow, estuarine waters with temperatures above 15 °C. [ABSTRACT FROM AUTHOR]

The neurotoxin β-N-methylamino-L-alanine (BMAA) has been identified as an environmental factor triggering neurodegenerative diseases such as Amyotrophic Lateral Sclerosis (ALS) and Alzheimer's disease (AD). We investigated the possible vectors of BMAA and its isomers 2,4-diaminobutyric acid (DAB) and N-2(aminoethyl)glycine (AEG) in marine mollusks collected from the Chinese coast. Sixty-eight samples of marine mollusks were collected along the Chinese coast in 2016, and were analyzed by an HILIC-MS/MS (hydrophilic interaction liquid chromatography with tandem quadrupole mass spectrometer) method without derivatization. BMAA was detected in a total of five samples from three species: Neverita didyma, Solen strictus, and Mytilus coruscus. The top three concentrations of free-form BMAA (0.99∼3.97 μg·g-1 wet weight) were detected in N. didyma. DAB was universally detected in most of the mollusk samples (53/68) with no species-specific or regional differences (0.051∼2.65 μg·g-1 wet weight). No AEG was detected in any mollusk samples tested here. The results indicate that the gastropod N. didyma might be an important vector of the neurotoxin BMAA in the Chinese marine ecosystem. The neurotoxin DAB was universally present in marine bivalve and gastropod mollusks. Since N. didyma is consumed by humans, we suggest that the origin and risk of BMAA and DAB toxins in the marine ecosystem should be further investigated in the future. [ABSTRACT FROM AUTHOR]

ß-N-methylamino-L-alanine (BMAA) is a neurotoxic non-protein amino acid suggested to be involved in neurodegenerative diseases. It was reported to be produced by cyanobacteria, but also found in edible aquatic organisms, thus raising concern of a widespread human exposure. However, the chemical analysis of BMAA and its isomers are controversial, mainly due to the lack of selectivity of the analytical methods. Using factorial design, we have optimized the chromatographic separation of underivatized analogues by a hydrophilic interaction chromatography coupled to tandem mass spectrometry (HILIC-MS/MS) method. A combination of an effective solid phase extraction (SPE) clean-up, appropriate chromatographic resolution and the use of specific mass spectral transitions allowed for the development of a highly selective and sensitive analytical procedure to identify and quantify BMAA and its isomers (in both free and total form) in cyanobacteria and mollusk matrices (LOQ of 0.225 and 0.15 |ig/g dry weight, respectively). Ten species of cyanobacteria (six are reported to be BMAA producers) were screened with this method, and neither free nor bound BMAA could be found, while both free and bound DAB were present in almost all samples. Mussels and oysters collected in 2009 in the Thau Lagoon, France, were also screened, and bound BMAA and its two isomers, DAB and AEG, were observed in all samples (from 0.6 to 14.4 μg/g DW), while only several samples contained quantifiable free BMAA. [ABSTRACT FROM AUTHOR]

Tetrodotoxins (TTXs), potent neurotoxins, have become an increasing concern in Europe in recent decades, especially because of their presence in mollusks. The European Food Safety Authority published a Scientific Opinion setting a recommended threshold for TTX in mollusks of 44 µg equivalent kg−1 and calling all member states to contribute to an effort to gather data in order to produce a more exhaustive risk assessment. The objective of this work was to assess TTX levels in wild and farmed mussels (Mytilus galloprovincialis) harvested in 2018–2019 along the coastal area of the Marche region in the Central Adriatic Sea (Italy). The presence of Vibrio spp. carrying the non-ribosomal peptide synthetase (NRPS) and polyketide synthase (PKS) genes, which are suspected to be involved in TTX biosynthesis, was also investigated. Out of 158 mussel samples analyzed by hydrophilic interaction liquid chromatography coupled with tandem mass spectrometry (HILIC-MS/MS), 11 (7%) contained the toxins at detectable levels (8–26 µg kg−1) and 3 (2%) contained levels above the EFSA safety threshold (61–76 µg kg−1). Contaminated mussels were all harvested from natural beds in spring or summer. Of the 2019 samples, 70% of them contained V. alginolyticus strains with the NRPS and/or PKS genes. None of the strains containing NRPS and/or PKS genes showed detectable levels of TTXs. TTXs in mussels are not yet a threat in the Marche region nor in Europe, but further investigations are surely needed. [ABSTRACT FROM AUTHOR]

Tetrodotoxin (TTX) is a potent neurotoxin, considered an emerging toxin in Europe where recently a safety limit of 44 µg TTX kg−1 was recommended by authorities. In this study, three specimens of the large gastropod trumpet shell Charonia lampas bought in a market in south Portugal were analyzed using a neuroblastoma cell (N2a) based assay and by LC-MS/MS. N2a toxicity was observed in the viscera of two individuals analyzed and LC-MS/MS showed very high concentrations of TTX (42.1 mg kg−1) and 4,9-anhydroTTX (56.3 mg kg−1). A third compound with m/z 318 and structurally related with TTX was observed. In the edible portion, i.e., the muscle, toxin levels were below the EFSA recommended limit. This study shows that trumpet shell marine snails are seafood species that may reach the markets containing low TTX levels in the edible portion but containing very high levels of TTX in non-edible portion raising concerns regarding food safety if a proper evisceration is not carried out by consumers. These results highlight the need for better understanding TTX variability in this gastropod species, which is critical to developing a proper legal framework for resources management ensuring seafood safety, and the introduction of these gastropods in the markets. [ABSTRACT FROM AUTHOR]

Tetrodotoxin (TTX) is a heat-stable neurotoxin typically associated with pufferfish intoxications. It has also been detected in shellfish from Japan, the United Kingdom, Greece, China, Italy, the Netherlands and New Zealand. A recent European Food Safety Authority (EFSA) scientific opinion concluded that a level of <0.044 mg TTX/kg in marine bivalves and gastropods, based on a 400 g portion size, does not result in adverse effects in humans. There have been no reports of human illness attributed to the consumption of New Zealand shellfish containing TTX. To obtain a greater understanding of its presence, a survey of non-commercial New Zealand shellfish was performed between December 2016 and March 2018. During this period, 766 samples were analysed from 8 different species. TTX levels were found to be low and similar to those observed in shellfish from other countries, except for pipi (Paphies australis), a clam species endemic to New Zealand. All pipi analysed as part of the survey were found to contain detectable levels of TTX, and pipi from a sampling site in Hokianga Harbour contained consistently elevated levels. In contrast, no TTX was observed in cockles from this same sampling site. No recreationally harvested shellfish species, including mussels, oysters, clams and tuatua, contained TTX levels above the recommended EFSA safe guidance level. The levels observed in shellfish were considerably lower than those reported in other marine organisms known to contain TTX and cause human intoxication (e.g., pufferfish). Despite significant effort, the source of TTX in shellfish, and indeed all animals, remains unresolved making it a difficult issue to understand and manage. [ABSTRACT FROM AUTHOR]

Paralytic shellfish toxins (PSTs) are the major neurotoxic contaminants of edible bivalves in Japan. Tetrodotoxin (TTX) was recently detected in bivalve shellfish around the world, drawing widespread attention. In Japan, high levels of TTX were reported in the digestive gland of the scallop, Patinopecten yessoensis, in 1993; however, no new data have emerged since then. In this study, we simultaneously analyzed PSTs and TTX in scallops cultured in a bay of east Japan using hydrophilic interaction chromatography (HILIC)-MS/MS. These scallops were temporally collected from April to December 2017. The highest concentration of PSTs (182 µmol/kg, total congeners) in the hepatopancreas was detected in samples collected on May 23, lined to the cell density of the dinoflagellate, Alexandrium tamarense, in seawater around the scallops, whereas the highest concentration of TTX (421 nmol/kg) was detected in samples collected on August 22. Contrary to the previous report, temporal variation of the PSTs and TTX concentrations did not coincide. The highest concentration of TTX in the entire edible tissues was 7.3 µg/kg (23 nmol/kg) in samples obtained on August 22, which was lower than the European Food Safety Authority (EFSA)-proposed threshold, 44 µg TTX equivalents/kg shellfish meat. In addition, 12β-deoxygonyautoxin 3 was firstly identified in scallops. [ABSTRACT FROM AUTHOR]

The neurotoxin β- N -methylamino-l-alanine (BMAA) has been identified as an environmental factor triggering neurodegenerative diseases such as Amyotrophic Lateral Sclerosis (ALS) and Alzheimer's disease (AD). We investigated the possible vectors of BMAA and its isomers 2,4-diaminobutyric acid (DAB) and N -2(aminoethyl)glycine (AEG) in marine mollusks collected from the Chinese coast. Sixty-eight samples of marine mollusks were collected along the Chinese coast in 2016, and were analyzed by an HILIC-MS/MS (hydrophilic interaction liquid chromatography with tandem quadrupole mass spectrometer) method without derivatization. BMAA was detected in a total of five samples from three species: Neverita didyma , Solen strictus , and Mytilus coruscus . The top three concentrations of free-form BMAA (0.99~3.97 μg·g -1 wet weight) were detected in N. didyma . DAB was universally detected in most of the mollusk samples (53/68) with no species-specific or regional differences (0.051~2.65 μg·g -1 wet weight). No AEG was detected in any mollusk samples tested here. The results indicate that the gastropod N. didyma might be an important vector of the neurotoxin BMAA in the Chinese marine ecosystem. The neurotoxin DAB was universally present in marine bivalve and gastropod mollusks. Since N. didyma is consumed by humans, we suggest that the origin and risk of BMAA and DAB toxins in the marine ecosystem should be further investigated in the future., Competing Interests: All authors declare no conflict of interest.

A novel bioanalytical method was developed and validated for the quantitative determination of erlotinib in human plasma by using the supported liquid extraction (SLE) sample cleanup coupled with hydrophilic interaction liquid chromatography and tandem mass spectrometric detection (HILIC-MS/MS). The SLE extract could be directly injected into the HILIC-MS/MS system for analysis without the solvent evaporation and reconstitution steps. Therefore, the method is simple and rapid. In the present method, erlotinib- d₆ was used as the internal standard. The SLE extraction recovery was 101.3%. The validated linear curve range was 2 to 2,000 ng/mL based on a sample volume of 0.100-mL, with a linear correlation coefficient of > 0.999. The validation results demonstrated that the present method gave a satisfactory precision and accuracy: intra-day CV < 5.9% (.