Search Results (25)

Gynecologic tumors are a leading cause of cancer-related mortality in women worldwide, with endometrial, ovarian, and cervical types being the most prevalent. Aberrant glycosylation, a key post-translational modification, plays a crucial role in tumor development, metastasis, and immune evasion. Specific glycosylation changes, such as altered sialylation and fucosylation, have been identified in gynecologic cancers and are associated with disease progression and prognosis. Understanding glycosylation alterations in gynecologic cancers holds promise for novel diagnostic and therapeutic approaches, ultimately enhancing patient outcomes. In this study, the serum N-glycome was analyzed in patients with myoma uteri, endometriosis, and cervical carcinoma by hydrophilic-interaction liquid chromatography (HILIC-UPLC) with fluorescence (FLR) and mass-spectrometric (MS) detection in order to identify their biomarker potential. Individual serum samples were deglycosylated by PNGase F digestion followed by procainamide labeling and solid-phase-extraction-based purification. All disease groups exhibited consistently higher levels of specific bi-antennary glycans (A2G2 and A2G2S1) compared to control patients. Additionally, significantly higher levels of agalactosylated and mono-sialylated glycans were found in cervical cancer, while a notable decrease in bisected N-glycans, alongside an increase in highly branched tetra-sialylated glycans, was found in endometriosis. Our study serves as proof-of-concept, demonstrating that discovering biomarkers within the serum N-glycome is a promising approach for identifying non-invasive indicators of gynecologic conditions. Full article

A simple method has been developed for the simultaneous analysis of ibuprofen (acid drug), and salbutamol and atenolol (basic drugs) in urine samples at concentrations of 0.40 µg·mL⁻¹. Simultaneous chromatographic separation has been possible using hydrophilic interaction liquid chromatography (Kinetex HILIC^® column (2.1 mm × 150 mm, 2.6 μm particle size diameter and 100 Å pore size) combined with gradient elution by employing a mixture of acetonitrile–acetate buffer 5 mM at pH 6 (from 95:5 to 75:25 (v/v)) as the mobile phase. Detection was performed at 227 and 275 nm. The simultaneous preconcentration and cleaning of the sample has been possible by solid-phase extraction using the HLB ExtraBond^® polymeric-type sorbent (which is a pyrrolidone-modified divinylbenzene polystyrene type). It has provided recoveries between (63 ± 9)% for salbutamol, (74 ± 8)% for ibuprofen, and (96 ± 9)% for atenolol in 10 mL of synthetic urine containing 4.0 μg of each of the drugs analyzed. The detection limits were 0.025 µg·mL⁻¹ for ibuprofen, µg·mL⁻¹ for salbutamol, and 0.007 µg·mL⁻¹ for atenolol. The detection limits obtained allow the evaluation of the free forms of ibuprofen, atenolol, and salbutamol at the excreted concentration levels at the therapeutic doses usually administered. The coefficients of variation between days were in the range 4.5–10.9%. Full article

The signature of human serum IgG glycosylation is critical in the defense against pathogens. Alterations of IgG N-glycome were associated with COVID-19 (Coronavirus disease 2019) severity, although knowledge on the response to vaccination is limited. IgG N-glycome was analyzed in this study in post-COVID-19 and post-vaccination patients to reveal potential glycosylation-based alterations using hydrophilic interaction liquid chromatography (HILIC-UPLC) with fluorescence (FLR) and mass-spectrometric (MS) detection. IgG antibodies were purified from serum samples through protein G affinity chromatography followed by PNGase F digestion-based deglycosylation. The released glycans were fluorescently derivatized by procainamide labeling and purified via solid-phase extraction. Higher levels of sialylation and afucosylation were identified in post-COVID-19 patients, which was further expanded by vaccination, but only in those who were previously SARS-CoV-2 (Severe acute respiratory syndrome coronavirus 2) infected. Full article

Τhyreostats (TSs) are veterinary drugs used in livestock farming for fattening. Their administration is banned in the European Union since 1981, and their monitoring for food quality and safety control requires sensitive and confirmatory methods. The present study describes the development and validation of a hydrophilic interaction liquid chromatography tandem mass spectrometry (HILIC-MS/MS) method for the simultaneous determination of 2-thiouracil (TU), 6-methyl-2-thiouracil (MTU), 6-propyl-2-thiouracil (PTU), 6-phenyl-2-thiouracil (PhTU), tapazole (TAP), and 2-mercaptobenzimidazole (MBI) in bovine muscle tissues. Investigation of the retention mechanism of the six analytes on the selected amide-based stationary phase showed that hydrophilic partition was the dominant interaction. The sample preparation included extraction with ACN/H₂O (80/20), followed by dispersive solid-phase extraction (d-SPE) with C18 sorbent and hexane partitioning. The method was validated according to European guidelines using internal standards, including isotopically labelled ones. The method’s LODs ranged between 2.8 ng g⁻¹ (6-phenyl-2-thiouracil) and 4.1 ng g⁻¹ (2-thiouracil). Application of the proposed method to 48 bovine tissue samples showed non-detectable results. Full article

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. Full article

Sample preparation for mass spectroscopy typically involves several liquid and solid phase clean-ups, extractions, and other unit operations, which are labour-intensive and error-prone. We demonstrate a centrifugal microfluidic platform that automates the whole blood sample’s preparation and clean-up by combining traditional liquid-phase and multiple solid-phase extractions for applications in mass spectroscopy (MS)-based small molecule detection. Liquid phase extraction was performed using methanol to precipitate proteins in plasma separated from a blood sample under centrifugal force. The preloaded solid phase composed of C18 beads then removed lipids with a combination of silica particles, which further cleaned up any remaining proteins. We further integrated the application of this sample prep disc with matrix-assisted laser desorption/ionization (MALDI) MS by using glancing angle deposition films, which further cleaned up the processed sample by segregating the electrolyte background from the sample salts. Additionally, hydrophilic interaction liquid chromatography (HILIC) MS was employed for detecting targeted free amino acids. Therefore, several representative ionic metabolites, including several amino acids and organic acids from blood samples, were analysed by both MALDI-MS and HILIC-MS to demonstrate the performance of this sample preparation disc. The fully automated blood sample preparation procedure only took 35 mins, with a throughput of three parallel units. Full article

Cytisine (CYT) is a quinolizidine alkaloid used for nicotine addiction treatment. Recent clinical trial data regarding cytisine confirm its high effectiveness and safety as a smoking cessation treatment. CYT’s popularity is growing due to its increased availability and licensing in more countries worldwide. This increased use by smokers has also resulted in an urgent need for continued drug research, including developing appropriate analytical methods for analyzing the drug in biological samples. In this study, a simple, fast, and reliable method combining hydrophilic interaction liquid chromatography and electrospray ionization quadrupole time of flight mass spectrometry (HILIC/ESI-QTOF-MS) for the determination of CYT in human serum and saliva was developed and validated. This was undertaken after the previous pre-treatment of the sample using solid-phase extraction (SPE). A hydrophilic interaction liquid chromatography (HILIC) column with a silica stationary phase was used for chromatographic analysis. In a linear gradient, the mobile phase consisted of acetonitrile (ACN) and formate buffer at pH 4.0. The proposed method was fully validated and demonstrated its sensitivity, selectivity, precision, and accuracy. The method was successfully applied to determine CYT in serum and, for the first time, in saliva. The findings indicate that saliva could be a promising non-invasive alternative to measure the free concentration of CYT. Full article

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. Full article

Kappa-casein-derived caseinomacropeptide (CMP)—a 64-amino-acid peptide—is released from kappa-casein after rennet treatment and is one of the major peptides in whey protein isolate (WPI). CMP has anti-inflammatory and antibacterial activities. It also has two major amino acid sequences with different modifications, including glycosylation, phosphorylation, and oxidation. To understand the potential biological role of CMP within the human body, there is a need to examine the extent to which CMP and CMP-derived fragments survive across the digestive tract, where they can exert these functions. In this study, three solid-phase extraction (SPE) methods—porous graphitized carbon (PGC), hydrophilic interaction liquid chromatography (HILIC), and C18 chromatography—were evaluated to determine which SPE sorbent is the most efficient to extract intact CMP and CMP-derived peptides from WPI and intestinal digestive samples prior to LC-MS/MS acquisition. The C18 SPE sorbent was the most efficient in extracting intact CMP and CMP-derived peptides from WPI, whereas the PGC SPE sorbent was the most efficient in extracting CMP-derived peptides from intestinal digesta samples. Full article

The optimization of solid-phase extraction (SPE) purification and chromatographic separation is usually neglected during proteomics studies. However, the effects on detection performance are not negligible, especially when working with highly glycosylated samples. We performed a comparative study of different SPE setups, including an in-house optimized method and reversed-phase chromatographic gradients for the analysis of highly glycosylated plasma fractions as a model sample for glycopeptide analysis. The in-house-developed SPE method outperformed the graphite-based and hydrophilic interaction liquid chromatography (HILIC) purification methods in detection performance, recovery, and repeatability. During optimization of the chromatography, peak distribution was maximized to increase the peptide detection rate. As a result, we present sample purification and chromatographic separation methods optimized for the analysis of hydrophilic samples, the most important of which is heavily N-glycosylated protein mixtures. Full article

Cyanobacteria are a diverse group of oxygenic photosynthetic prokaryotes, which are believed to be one of the oldest life forms on Earth. They live in a wide range of ecosystems and withstand extreme environmental conditions. An important proportion of cyanobacteria is known to be producers of harmful cyanotoxins, which are toxic secondary metabolites that can impact the ecosystem and human health. The oral route is one of the main ways whereby humans can be exposed to cyanotoxins. Therefore, the consumption of contaminated algae-based food supplements is becoming more relevant due to its upsurge, which underlines the importance of controlling these toxins in this kind of products. This work describes the simultaneous determination of seven cyanotoxins belonging to three different classes: the cyclic peptides microcystin-LR (MC-LR), microcystin-RR (MC-RR) and nodularin (NOD); the alkaloid anatoxin-a (ANA) and three non-protein amino acids isomers β-methylamine-L-alanine (BMAA), 2,4-diaminobutyric acid (DAB) and N-(2-aminoethyl)glycine (AEG). These have been determined in spirulina-derived food supplements using a novel solid–liquid extraction coupled with a solid phase extraction procedure for clean up and preconcentration (SLE-tandem-SPE) and analysis by hydrophilic interaction liquid chromatography with tandem mass spectrometry detection (HILIC-MS/MS). A SeQuant® Zwitterionic Hydrophilic Interaction Liquid Chromatography (ZIC-HILIC) column (EMD Millipore, Billerica, MA, USA) was employed to achieve the chromatographic separation in less than 12 min using water and acetonitrile, both acidified with 0.3% of formic acid, as mobile phase. Previously, an SLE was developed, using 4 mL of aqueous 5% formic acid to extract the most polar compounds, followed by 4 mL of 80% MeOH. Both extracts were combined and submitted to a tandem-SPE using mixed-mode cation exchange (MCX) and Strata-X cartridges. Elution from both cartridges was performed using 10% NH₃·H₂O in MeOH. Method validation was carried out in terms of linearity, limit of detection (LOD) and quantification (LOQ), recoveries, matrix effect and repeatability and intermediate precision. LOQs in the range of 50–300 µg·kg⁻¹ and recoveries ranging between 64.2% and 102.9% with an associated relative standard deviation < 19.2% were achieved. Satisfactory precision was obtained with RSD values lower than 19.6% in all cases, with the exception of BMAA, which reported the highest RSD values, reaching 25.1%. The method was satisfactorily applied to determine the occurrence of cyanotoxins in blue green algae (BGA) dietary supplements. DAB was the most frequently detected cyanotoxin, at concentrations up to 2408 µg·kg⁻¹, and AEG was found in few samples at concentrations up to 194 µg·kg⁻¹. However, MC-LR and MC-RR were found in one sample at concentration levels higher than 5 mg·kg⁻¹, which underlines the need to control these substances in these matrices. Full article

Neurotoxin β-N-methylamino-L-alanine (BMAA) is hypothesized as an important pathogenic factor for neurodegenerative diseases such as amyotrophic lateral sclerosis/parkinsonism-dementia complex (ALS-PDC). Comparative study on the accuracy of BMAA analyzed by the regular LC-MS/MS methods is still limited for different biological matrices. In this study, a free-BMAA sample of cyanobacterium and BMAA-containing positive samples of diatom, mussel, scallop, and oyster were extracted with varied extraction ratios (ER) ranging from 1:20 to 1:2000. These extracts were then purified by MCX cartridges. After SPE purification, these different biological samples were analyzed by two common LC-MS/MS analysis methods, a direct analysis without derivatization by a hydrophilic interaction liquid chromatography (HILIC)-MS/MS and pre-column 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate (AQC) derivatization combined with a C18 column. The results suggested that the recoveries of BMAA spiked in the cyanobacterial sample were close to 100% in the total soluble form extracts with the ER of 1:100 (g/mL) and the precipitated bound form extracts with the ER of 1:500. The recommended ER for the precipitated bound form of BMAA in diatoms and the total soluble form of BMAA in mollusks are 1:500 and 1:50, respectively. The quantitative results determined by the AQC derivatization method were lower than those determined by the direct analysis of the HILIC method in diatom and mollusk samples. The results of the HILIC method without the derivatization process were closer to the true value of BMAA in cyanobacteria. This work contributes to the performance of the solid-phase extraction (SPE) purification protocol and the accuracy of BMAA analysis by LC-MS/MS in diverse biological samples. Full article

Olive seeds have been considered as a new nutritionally healthy food supplement. They are rich in monounsaturated n-9 and essential polyunsaturated n-6 lipids. However, little is known about their polar lipids, potentially bioactive and chemical identity markers for olive pulp and oil. This work aimed to identify the polar lipidome of olive seeds to find possible bioactive compounds and markers of geographic origin, by studying samples from six Portuguese sub-regions. Polar lipids were obtained by solid/liquid extraction, NH₂-solid-phase extraction, and identified by hydrophilic interaction liquid chromatography (HILIC)-HR-ESI-MS and MS/MS. Ninety-four compounds were identified, including phospholipids, glycolipids, sphingolipids, and acyl sterol glycosides, several of which bear polyunsaturated fatty acids. Multivariate statistical analysis found unique profiles within each sub-region and markers of geographic identity, primarily phosphatidylcholines, phosphatidylethanolamines, and lysophosphatidylethanolamines. Therefore, polar lipid signatures should be further investigated, to assess their bioactivity, nutritional value, and chemical identity for valuing olive seeds and their oil. Full article

A rapid, sensitive method for the separation of catecholamine biomarkers (CAs), of importance in traumatic brain injury (TBI) and in Parkinson’s disease (PD), has been successfully developed using hydrophilic interaction liquid chromatography (HILIC). Dopamine (DA), epinephrine (EPI), and norepinephrine (NE) are known to be three to fivefold elevated above normal in traumatic brain injury (TBI) patients. HILIC facilitates the rapid and efficient separation of these polar biomarkers, which can be poorly retained by reversed-phase liquid chromatography (RPLC), while electrochemical detection (ECD) at the boron-doped diamond (BDD) electrode provides enhanced nanomolar detection. Three HILIC columns were compared, namely the superficially porous (core-shell) Z-HILIC column and the Z-cHILIC and Z-HILIC fully porous columns. The core-shell Z-HILIC showed the highest efficiency with a rapid separation within 60 s. The HILIC method utilizing the core-shell Z-HILIC column was initially optimized for the simultaneous analysis of DA, EPI, and NE using UV detection. The advantages of using the BDD electrode over UV detection were explored, and the improved limits of detection (LODs, S/N = 3) measured were 40, 50, and 50 nM for DA, EPI, and NE, respectively. Method validation is reported in terms of the linearity, repeatability, reproducibility, and LODs. Furthermore, the proposed method was successfully applied to the real sample analysis of urinary CAs following phenylboronic acid (PBA) solid phase extraction (SPE) pretreatment. Full article

Biochemical methylation reactions mediate the transfer of the methyl group regulating vital biochemical reactions implicated in various diseases as well as the methylation of DNA regulating the replication processes occurring in living organisms. As a finite number of methyl carriers are involved in the methyl transfer, their quantification could aid towards the assessment of an organism’s methylation potential. An Hydrophilic Interaction Chromatography-Liquid Chromatography Multiple Reaction Monitoring (HILIC-LC-MRM) mass spectrometry (MS) methodology was developed and validated according to Food & Drug Administration (FDA), European Medicines Agency (EMA), and International Council for Harmonisation of Technical Requirements for Pharmaceuticals for Human Use (ICH) for the simultaneous determination of nine metabolites i.e., B12, folic acid, 5-methyltetrahydrofolate, S-adenosylmethionine, S-adenosylhomocysteine, betaine, phosphocholine, N,N-dimethylglycine, and deoxythymidine monophosphate in human blood plasma. The sample pretreatment was based on a single step Solid-phase extraction (SPE) methodology using C18 cartridges. The methodology was found to accurately quantitate the analytes under investigation according to the corresponding dynamic range proposed in the literature for each analyte. The applicability of the method was assessed using blood donor samples and its applicability demonstrated by the assessment of their basal levels, which were shown to agree with the established basal levels. The methodology can be used for diagnostic purposes as well as for epigenetic screening. Full article