Research

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17 pages, 5449 KiB

Open AccessArticle

Urinary Proteomic/Peptidomic Biosignature of Breast Cancer Patients Using 1D SDS-PAGE Combined with Matrix-Assisted Laser Desorption/Ionization-Time of Flight Mass Spectrometry

by Patrícia Sousa, Irene Camacho, José S. Câmara and Rosa Perestrelo

Separations 2023, 10(5), 291; https://0-doi-org.brum.beds.ac.uk/10.3390/separations10050291 - 05 May 2023

Cited by 3 | Viewed by 1627

Abstract

The potential development of a rapid and highly sensitive breast cancer (BC) diagnostic method has been increasingly investigated by many researchers in order to significantly improve the diagnosis of this disease that affects millions of women worldwide. Thus, this investigation aimed to establish [...] Read more.

The potential development of a rapid and highly sensitive breast cancer (BC) diagnostic method has been increasingly investigated by many researchers in order to significantly improve the diagnosis of this disease that affects millions of women worldwide. Thus, this investigation aimed to establish a potential BC urinary peptidomic pattern using one-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis (1D SDS-PAGE) coupled with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) as a useful approach for BC diagnosis. The results of Lowry’s assay demonstrated that the total protein concentration increased after precipitation and that the healthy control group (HCs, 160 to 318 µg/mL, 142 ± DD µg/mL, on average) presented higher total protein content than the BC patients (140 to 311 µg/mL, 115 ± DD µg/mL, on average). Related to MALDI-TOF MS analysis, the results revealed that four peptide ion biosignatures (m/z 1046.5, 1062.5, 1237.7 and 1727.9) allowed the discrimination between BC patients and HCs. The distinction efficiency and accuracy of BC urine peptides were determined by receiver operating characteristic (ROC) curve analysis that enabled the recognition of some features with great sensitivity (88%) and specificity (98%). Therefore, the obtained data revealed MALDI-TOF MS as a powerful tool to explore peptidomic biosignatures due to its speed, sensitivity, and mass accuracy, which allow the establishment of novel disease biomarkers. Full article

(This article belongs to the Special Issue Chromatographic and Electrophoretic Methods in Current Biomedical Analysis)

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13 pages, 3362 KiB

Open AccessArticle

Simple and Sensitive Analysis of Clenbuterol in Urine Matrices by UHPLC-MS/MS Method with Online-SPE Sample Preparation

by Kristián Slíž, Dominika Olešová, Juraj Piešťanský and Peter Mikuš

Separations 2022, 9(12), 440; https://0-doi-org.brum.beds.ac.uk/10.3390/separations9120440 - 14 Dec 2022

Cited by 2 | Viewed by 1904

Abstract

Clenbuterol is one of the most misused anabolic agents in professional sports. Therefore, the monitoring of clenbuterol in body fluids such as human urine is related to the development of rapid, selective and sensitive analytical methods that produce reliable results. In this work, [...] Read more.

Clenbuterol is one of the most misused anabolic agents in professional sports. Therefore, the monitoring of clenbuterol in body fluids such as human urine is related to the development of rapid, selective and sensitive analytical methods that produce reliable results. In this work, these requirements were met by a two-dimensional separation method based on online solid-phase extraction coupled with ultra-high performance liquid chromatography–tandem mass spectrometry (SPE–UHPLC–MS/MS). The developed method provides favorable performance parameters, and it is characterized by minimum manual steps (only dilution and the addition of an internal standard) in the sample preparation. A limit of quantification (LOQ) of 0.1 ng/mL, excellent linearity (0.9999), remarkable precision (1.26% to 8.99%) and high accuracy (93.1% to 98.7%) were achieved. From a practical point of view, the analytical performance of the validated SPE–UHPLC–MS/MS method was demonstrated on blinded spiked urine samples from ten healthy volunteers. The estimated concentrations of clenbuterol were in accordance with their corresponding nominal values, as supported by the precision and accuracy data (relative standard deviation ≤5.4%, relative error ≤11%). The fulfillment of the World Anti-Doping Agency’s screening and confirmation criteria indicates that the proposed method is suitable for implementation in routine use in toxicologic and antidoping laboratories. Due to its high orthogonality and separation efficiency, the SPE–UHPLC–MS/MS method should also be easily adapted to the separation of structurally related compounds (such as clenbuterol metabolites). Thus, future antidoping applications could also include monitoring of clenbuterol metabolites, providing a longer detection widow. Full article

(This article belongs to the Special Issue Chromatographic and Electrophoretic Methods in Current Biomedical Analysis)

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8 pages, 1193 KiB

Open AccessArticle

Validated Capillary Zone Electrophoresis Method for Impurity Profiling and Determination of Ni^II(3-OMe-Salophene)

by Sami El Deeb, Adel Ehab Ibrahim, Ahmed Al-Harrasi, Gerhard Wolber and Ronald Gust

Separations 2022, 9(2), 25; https://0-doi-org.brum.beds.ac.uk/10.3390/separations9020025 - 21 Jan 2022

Cited by 5 | Viewed by 2272

Abstract

A capillary zone electrophoresis method was developed for the determination of Ni^II(3-OMe-salophene), a substance with anticancer activity in vitro. A fused silica capillary (56 cm × 100 µm) was used for this purpose. The method was optimized in terms of parameters [...] Read more.

A capillary zone electrophoresis method was developed for the determination of Ni^II(3-OMe-salophene), a substance with anticancer activity in vitro. A fused silica capillary (56 cm × 100 µm) was used for this purpose. The method was optimized in terms of parameters affecting the electrophoretic conditions in order to optimize separation efficiency and total time of migration. The analysis was best performed using an operating buffer of 50 mM borate, adjusted to pH 9.3, mixed with acetonitrile (50%, v/v) as organic modifier. Injections were performed hydrodynamically by applying a pressure of 50 mbar for 8 s, and a 30 kV separation voltage was selected at 25 °C. Detection was carried out at 250 nm using diode array detector (DAD). The method allowed the separation of Ni^II(3-OMe-salophene) from four other structurally related impurities in a total migration time (t_m) of 8 min. Peak identification was achieved using the standard reference of individual impurities. The purity of the migrated Ni^II(3-OMe-salophene) was confirmed by Ultra-violet (UV) scan overlay depending on DAD. The linear ranges for the determination of Ni^II(3-OMe-salophene) was 400–20,000 ng mL⁻¹ with limit of detection (LOD) of 120 ng mL⁻¹. Acceptable intra-day and inter-day precisions were achieved (%relative standard deviation (RSD) results were less than 0.76% and 0.30%, respectively). The proposed method was assessed for greenness and compared to reported methodologies to prove superiority. Full article

(This article belongs to the Special Issue Chromatographic and Electrophoretic Methods in Current Biomedical Analysis)

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14 pages, 1996 KiB

Open AccessArticle

Targeted UHPLC-ESI-MS/MS Analysis of Selected Neurotransmitters, Tryptophan and Its Metabolite Kynurenine in Tau Transgenic Rat Brain Tissue: A Pivotal Study

by Juraj Piestansky, Andrea Forgacsova, Dominika Olesova, Jaroslav Galba, Peter Mikus, Petra Majerova and Andrej Kovac

Separations 2022, 9(1), 16; https://0-doi-org.brum.beds.ac.uk/10.3390/separations9010016 - 14 Jan 2022

Cited by 3 | Viewed by 2594

Abstract

Neurotransmitters (NT) are widely distributed in the central nervous system. These molecules are important for many physiological processes and the function of the immune system. Imbalance of NT are linked to numerous neurological disorders and diseases, including tauopathies. Here, a targeted approach based [...] Read more.

Neurotransmitters (NT) are widely distributed in the central nervous system. These molecules are important for many physiological processes and the function of the immune system. Imbalance of NT are linked to numerous neurological disorders and diseases, including tauopathies. Here, a targeted approach based on on-line combination of ultra-high performance liquid chromatography with tandem mass spectrometry was validated and applied to the quantitative analysis of nine NT (acetylcholine, choline, aspartic acid, asparagine, glutamic acid, glutamine, pyroglutamate, γ-aminobutyric acid, N-acetyl-L-aspartic acid), tryptophan and its metabolite kynurenine in brain tissue samples of a rat model for tauopathy. The applied analytical method was characterized by excellent validation parameters for all analytes, such as limits of detection in the range of 0.01–1.70 µg/mL, regression coefficients of the calibration curves ≥ 0.9946, intra-day and inter-day precision expressed as coefficient of variation in the range of 0.6–11.9% and 0.6–14.4%, and accuracy in the range of 87.6–107.1% and 87.2–119.6%. Our analytical approach led to the identification of increased levels of choline and γ-aminobutyric acid in pons, and elevated concentration levels of pyroglutamate in medulla oblongata. These findings indicate that NT could play a valuable role in the study and clarification of neuroinflammation and neurodegenerative diseases. Full article

(This article belongs to the Special Issue Chromatographic and Electrophoretic Methods in Current Biomedical Analysis)

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9 pages, 4796 KiB

Open AccessFeature PaperEditor’s ChoiceArticle

Green Stability Indicating Organic Solvent-Free HPLC Determination of Remdesivir in Substances and Pharmaceutical Dosage Forms

by Adel Ehab Ibrahim, Sami El Deeb, Emad Mahmoud Abdelhalim, Ahmed Al-Harrasi and Rania Adel Sayed

Separations 2021, 8(12), 243; https://0-doi-org.brum.beds.ac.uk/10.3390/separations8120243 - 12 Dec 2021

Cited by 21 | Viewed by 4295

Abstract

A green liquid chromatographic method is considered in this work to minimize the environmental impact of waste solvents. One important principle is to replace or eliminate the use of hazardous organic solvents. Organic impurities in any active pharmaceutical ingredient could arise either during [...] Read more.

A green liquid chromatographic method is considered in this work to minimize the environmental impact of waste solvents. One important principle is to replace or eliminate the use of hazardous organic solvents. Organic impurities in any active pharmaceutical ingredient could arise either during the process of its synthesis, or as degradation products developed throughout the shelf-life. Remdesivir (RDS) is an antiviral drug, approved by the US Food and Drug Adminstration (-FDA), to treat SARS-Cov-2 virus during its pandemic crisis. We studied the stability of remdesivir against several degradation pathways using the organic solvent-free liquid chromatographic technique. Separation was performed on RP-C18 stationary phase using mixed-micellar mobile phase composed of a mixture of 0.025 M Brij-35, 0.1 M sodium lauryl sulfate (SLS), and 0.02 M disodium hydrogen phosphate, adjusted to pH 6.0. The mobile phase flow rate was 1 mL min⁻¹, and detection was carried out at a wavelength of 244 nm. We profiled the impurities that originated in mild to drastic degradation conditions. The method was then validated according to International Conference of Harmonization (ICH) guidelines within a linearity range of 5–100 μg mL⁻¹ and applied successfully for the determination of the drug in its marketed dosage form. A brief comparison was established with reported chromatographic methods, including a greenness assessment on two new metrics (GAPI and AGREE). This study is the first to be reported as eco-friendly, solvent-free, and stability indicating LC methodology for RDS determination and impurity profiling. Full article

(This article belongs to the Special Issue Chromatographic and Electrophoretic Methods in Current Biomedical Analysis)

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13 pages, 1832 KiB

Open AccessArticle

Plasma Short-Chain Fatty Acids and Their Derivatives in Women with Gestational Diabetes Mellitus

by Eliška Ivanovová, Barbora Piskláková, Jaroslava Friedecká, Ondřej Krystyník, David Friedecký and David Karásek

Separations 2021, 8(10), 188; https://0-doi-org.brum.beds.ac.uk/10.3390/separations8100188 - 14 Oct 2021

Cited by 2 | Viewed by 2705

Abstract

Gestational diabetes mellitus (GDM) represents a heterogeneous group of hyperglycemic metabolic disorders that are associated with health outcomes for mothers and offspring. Currently, diagnosis of GDM is based on repetitive measurement of increased fasting plasma glucose (FPG) or upon results showing increased postprandial [...] Read more.

Gestational diabetes mellitus (GDM) represents a heterogeneous group of hyperglycemic metabolic disorders that are associated with health outcomes for mothers and offspring. Currently, diagnosis of GDM is based on repetitive measurement of increased fasting plasma glucose (FPG) or upon results showing increased postprandial plasma glucose (PPG). Recently, it was discovered that the changes in the gut microbiome during pregnancy are associated with insulin resistance and obesity. Therefore, in this study, relevant products of gut bacteria, short-chain fatty acids (SCFA) and their derivatives were evaluated together with baseline body composition characteristics and common biochemical parameters in women with three different phenotypes of GDM, healthy pregnant and nonpregnant women. Plasma SCFA and their derivatives were derivatized, separated on reversed-phase liquid chromatography and detected by a triple-quadrupole mass spectrometer. 3-hydroxybutyrate (3-OH-BA), 4-methylvalerate (4-MVA) and isovalerate (IVA), together with selected parameters associated with baseline body composition characteristics and biochemistry, were evaluated as statistically significant. 3-OH-BA, which was increased in all three groups of women with different phenotypes of GDM, reflects a ketogenic state of GDM. In all groups of pregnant women, elevated/suppressed concentrations of 4-MVA/IVA were found. These findings show the importance of monitoring SCFA and other parameters besides glucose in women with GDM. Full article

(This article belongs to the Special Issue Chromatographic and Electrophoretic Methods in Current Biomedical Analysis)

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8 pages, 1029 KiB

Open AccessFeature PaperArticle

Uncertainty of Size-Exclusion Chromatography Method in Quality Control of Bevacizumab Batches

by Alexis Oliva and Matías Llabrés

Separations 2021, 8(9), 133; https://0-doi-org.brum.beds.ac.uk/10.3390/separations8090133 - 25 Aug 2021

Cited by 2 | Viewed by 1871

Abstract

In addition to the analytical challenges related to the size and complexity of biopharmaceutical drugs, the inherent variability that arises due to their manufacturing process requires monitoring throughout the production process to ensure the safety and efficacy of the finished product. In this [...] Read more.

In addition to the analytical challenges related to the size and complexity of biopharmaceutical drugs, the inherent variability that arises due to their manufacturing process requires monitoring throughout the production process to ensure the safety and efficacy of the finished product. In this step, validation data should demonstrate that the process is controlled and reproducible, whereas the manufacturing process must ensure the quality and consistency of the product. For this, the manufacturer sets specification limits according with regulatory guidance. In such a situation, the comparison of different batches is required in order to describe and analyze the variability between them. However, it is unclear how great the variability of the analytical method would be or that in producing the batches. The estimation of the β-expectation tolerance intervals based on the variance components to account for both between-batch and within-batch variability was proposed as a specification limit to control the heterogeneity between batches at the time of manufacture and to verify whether batches meet specification limits. At this point, the variance components were computed by the maximum likelihood method using a linear random model. For this, the protein content, expressed as a percentage of the actual concentration relative to the claim value, and the dimer content (expressed as percentage) were used as critical quality attributes (CQAs) in the monitoring and control process. We used real data from six bevacizumab commercial batches. Full article

(This article belongs to the Special Issue Chromatographic and Electrophoretic Methods in Current Biomedical Analysis)

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Review

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36 pages, 2901 KiB

Open AccessReview

Recent Strategies for Using Monolithic Materials in Glycoprotein and Glycopeptide Analysis

by Allan J. Alla and Keith J. Stine

Separations 2022, 9(2), 44; https://0-doi-org.brum.beds.ac.uk/10.3390/separations9020044 - 05 Feb 2022

Cited by 7 | Viewed by 3647

Abstract

There is continuous effort towards developing monolithic materials as solid supports for the separation, enrichment, and digestion of glycoproteins. The intention of this review is to discuss and summarize work reported in this area during the period 2015–2021 as a follow-up to our [...] Read more.

There is continuous effort towards developing monolithic materials as solid supports for the separation, enrichment, and digestion of glycoproteins. The intention of this review is to discuss and summarize work reported in this area during the period 2015–2021 as a follow-up to our prior review. Reports from the past three decades have already proven the advantages of monolithic materials, such as the ease with which they can be prepared and functionalized, their high permeability and low resistance to mass transfer, and their stability over a wide range of pH. Recent works on glycoprotein analysis introduce different strategies in using monolithic materials specifically in separation, enrichment, and identification of glycoproteins, glycopeptides, and free glycans. A majority of these are focused on boronic acid affinity-based technique and others on lectin affinity and HILIC-based techniques. There are also newly developed ligands that utilize different interactions with glycans, such as encapsulation into β-cyclodextrin vesicles, CH- or OH-π interactions with fullerenes, immunoaffinity with monoclonal antibodies, H-bonding interactions with metallophthalocyanines, coordination interactions with cobalt phthalocyanine tetracarboxylic acid, and hydrophilic interaction with cyclodextrin molecular tubes, zwitterionic iminodiacetic acid, and boric acid. Recent strategies for developing on-line, multidimensional systems use immobilized monolithic enzyme reactors (IMERs) for high-throughput glycoprotein analysis. These works serve as contributions to better understand glycan structure-function relationship, as glycoproteins are now widely accepted disease biomarkers. Full article

(This article belongs to the Special Issue Chromatographic and Electrophoretic Methods in Current Biomedical Analysis)

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