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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.

A sensitive and selective hydrophilic interaction liquid chromatography-tandem mass spectrometry (HILIC-MS/MS) method was developed and validated for trace analysis of potential genotoxic impurities (PGIs): 2,3-dichloroaniline (PGI-1), bis(2-chloroethyl) amine (PGI-2), and 2-chloroethylamine (PGI-3), in aripiprazole (APZ) active drug substance. Separation of analytes was achieved on ACE HILIC–N Column (HILN-5-1046U, 100 × 4.6 mm, 5 μm) in gradient elution mode with mobile phase A [acetonitrile:ammonium formate buffer (95:5 v/v)] and mobile phase B [acetonitrile:ammonium formate buffer (50:50 v/v)] at a flow rate of 0.8 mL/min. Developed method was linear in the concentration range of 8–100 ppm for PGI-1, 11–100 ppm for PGI-2, and 12.5–100ppm for PGI-3 with R2 > 0.996. The developed method was accurate for quantification of each PGI with percent recoveries greater than 96% and RSD (%) not more than 5%. The developed method was precise for quantification of PGIs in aripiprazole with RSD (%) of not more than 4% for any of the PGIs. There was no interference of diluent peaks at the retention time of the PGIs and APZ in the method. All the PGIs and sample solutions were found to be stable at ambient laboratory temperature (25 ± 5 °C) and refrigerated condition (2–8 °C) for a period of 48 h. The developed HILIC-MS/MS method can be used for trace quantification of PGIs in aripiprazole drug in quality control laboratories of the pharmaceutical industry.

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.

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.

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 &lt, 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.

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.

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.

Monitoring carnitine and acetylcarnitine levels in biological fluids is a powerful tool for diagnostic studies. Research has recently shown that the analysis of carnitine and related compounds in clinical samples can be accomplished by different analytical approaches. Because of the polar and ionic nature of the analytes and matrix complexity, accurate quantitation is a highly challenging task. Thus, sample processing factors, preparation/cleanup procedures, and chromatographic/ionization/detection parameters were evaluated. On the basis of the results obtained, a rapid, selective, sensitive method based on hydrophilic interaction liquid chromatography?tandem mass spectrometry for the analysis of carnitine and acetylcarnitine in serum and urine samples is proposed. The matrix effect was assessed. The proposed approach was validated, the limits of detection were in the nanomolar range, and carnitine and acetylcarnitine levels were found within the micromolar range in both types of sample. Fil: Isaguirre, Andrea Celeste. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico San Luis. Instituto de Química de San Luis; Argentina Fil: Olsina, Roberto Antonio. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico San Luis. Instituto de Química de San Luis; Argentina Fil: Martinez, Luis Dante. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico San Luis. Instituto de Química de San Luis; Argentina Fil: Lapierre, Alicia V.. Universidad Nacional de San Luis. Departamento de Qui­mica; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico San Luis. Instituto de Química de San Luis; Argentina Fil: Cerutti, Estela Soledad. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico San Luis. Instituto de Química de San Luis; Argentina

Objectives: The aim of this work was to implement a fast, accurate and simple method to quantify plasma ADMA and SDMA, in a run time suitable for routine analysis. Design and methods: We developed and validated a hydrophilic interaction chromatographic method coupled to tandem mass spectrometry (HILIC-MS/MS) for separation and simultaneous quantification of Arginine (Arg) and its dimethylarginines, ADMA and SDMA, with a short run time (less than 5min) using a small volume of human plasma (0.02mL). Results: Correlation coefficients (r) of the calibration curves ranged from 0.9926 to 0.9984. Within-day and between-day imprecision (CV%) and inaccuracy (%), carry-over and recovery were also evaluated for validation. Preliminary data of Arg, ADMA and SDMA from 30 apparently healthy subjects and type 2 diabetic patients (n = 33) with and without kidney dysfunction were calculated and some statistical differences occurred among them (p < 0.05). Conclusions: Data from calibration curves and quality controls reveal that the method is accurate and precise. Healthy subjects and diabetic patients' values are in agreement with those reported in other studies. © 2008 The Canadian Society of Clinical Chemists

Reversed phase liquid chromatography (RPLC) coupled to mass spectrometry (MS) is the gold standard technique in bioanalysis. However, hydrophilic interaction chromatography (HILIC) could represent a viable alternative to RPLC for the analysis of polar and/or ionizable compounds, as it often provides higher MS sensitivity and alternative selectivity. Nevertheless, this technique can be also prone to matrix effects (ME). ME are one of the major issues in quantitative LC-MS bioanalysis. To ensure acceptable method performance (i.e., trueness and precision), a careful evaluation and minimization of ME is required. In the present study, the incidence of ME in HILIC-MS/MS and RPLC-MS/MS was compared for plasma and urine samples using two representative sets of 38 pharmaceutical compounds and 40 doping agents, respectively. The optimal generic chromatographic conditions in terms of selectivity with respect to interfering compounds were established in both chromatographic modes by testing three different stationary phases in each mode with different mobile phase pH. A second step involved the assessment of ME in RPLC and HILIC under the best generic conditions, using the post-extraction addition method. Biological samples were prepared using two different sample pre-treatments, i.e., a non-selective sample clean-up procedure (protein precipitation and simple dilution for plasma and urine samples, respectively) and a selective sample preparation, i.e., solid phase extraction for both matrices. The non-selective pretreatments led to significantly less ME in RPLC vs. HILIC conditions regardless of the matrix. On the contrary, HILIC appeared as a valuable alternative to RPLC for plasma and urine samples treated by a selective sample preparation. Indeed, in the case of selective sample preparation, the compounds influenced by ME were different in HILIC and RPLC, and lower and similar ME occurrence was generally observed in RPLC vs. HILIC for urine and plasma samples, respectively. The complementary of both chromatographic modes was also demonstrated, as ME was observed only scarcely for urine and plasma samples when selecting the most appropriate chromatographic mode.

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.

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.

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% (