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Separations
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Application of Hyphenated Techniques in Bioanalysis and Metabolite Identification
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Application of Hyphenated Techniques in Bioanalysis and Metabolite Identification

A special issue of Separations (ISSN 2297-8739). This special issue belongs to the section "Chromatographic Separations".

Deadline for manuscript submissions: closed (10 February 2024) | Viewed by 10590

Special Issue Editors

Prof. Dr. Rasha S. Hanafi

E-Mail Website
Guest Editor
Department of Pharmaceutical Chemistry, Faculty of Pharmacy and Biotechnology, The German University in Cairo, New Cairo 11835, Egypt
Interests: hyphenated LC and GC with mass spectrometry; metabolomics and biomarker discovery; pharmaceutical and bioanalysis; Quality by Design; chemometrics
Prof. Dr. Mohamed Z. Gad

E-Mail Website
Guest Editor
Department of Biochemistry, Faculty of Pharmacy and Biotechnology, The German University in Cairo, New Cairo 11835, Egypt
Interests: metabolomics and biomarker discovery; gasotransmitters; approaches for biomarkers assessment in body fluids; interpretation of metabolite profiling and its correlation to biochemical pathways

Special Issue Information

Dear Colleagues,

This Special Issue is dedicated to research and review articles covering the separation, identification, characterization and determination of metabolites and molecules generated in biological systems, including tissues and biofluids of human and animal origin, as well as in microorganisms and plants. The Issue also covers metabolomics and metabolite profiling using targeted or untargeted approaches that aim to estimate the variable metabolic responses of living systems under normal and pathological conditions. It is well-known that, due to the complexity of the metabolome or generated molecules in a living system, and due to the challenges in bioanalysis including sample extraction and preparation, no single analytical method can be used to analyze all metabolites in a biological sample. Accordingly, this Special Issue focuses principally on the application of liquid and gas chromatography-mass spectrometry (GC-MS and LC-MS) or high-resolution mass spectrometry in the analysis of biological samples as well as innovative sample preparation techniques, aiming to provide a good collection of novel articles to solve bioanalytical challenges and unravel the mystery of metabolite profiling in health and disease in humans and other species.  

Prof. Dr. Rasha S. Hanafi
Prof. Dr. Mohamed Z. Gad
Guest Editors

Manuscript Submission Information

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Submitted manuscripts should not have been published previously, nor be under consideration for publication elsewhere (except conference proceedings papers). All manuscripts are thoroughly refereed through a single-blind peer-review process. A guide for authors and other relevant information for submission of manuscripts is available on the Instructions for Authors page. Separations is an international peer-reviewed open access monthly journal published by MDPI.

Please visit the Instructions for Authors page before submitting a manuscript. The Article Processing Charge (APC) for publication in this open access journal is 2600 CHF (Swiss Francs). Submitted papers should be well formatted and use good English. Authors may use MDPI's English editing service prior to publication or during author revisions.

Keywords

  • metabolites
  • liquid chromatography
  • metabolomics
  • qualitative analysis
  • biological samples
  • human
  • plant
  • microorganism
  • separation
  • mass spectrometry
  • gas chromatography
  • bioanalysis
  • sample treatment

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Published Papers (5 papers)

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Research

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19 pages, 4864 KiB  
Article
Applicable Pharmacokinetic Study: Development and Validation of Bioanalytical LC-MS/MS Method for Simultaneous Determination of Tipiracil, Trifluridine and Its Two Metabolites 5-Trifluoromethyluracil, 5-Carboxy 2′-Deoxyuridine in Rat Plasma
by Manal El-Gendy, Mohamed Hefnawy, Adeeba Alzamil, Adel El-Azab, Alaa Abdel-Aziz and Ali El Gamal
Separations 2024, 11(1), 10; https://doi.org/10.3390/separations11010010 - 26 Dec 2023
Viewed by 1909
Abstract
A novel liquid chromatography-tandem mass spectrometry (LC-MS/MS) method has been developed and validated for the simultaneous determination of tipiracil (TIP), trifluridine (FTD), and their metabolites, 5-trifluoromethyluracil (FTY) and 5-carboxy-2′-deoxyuridine (5CDU), in rat plasma. This method is highly sensitive, specific, and fast. Paracetamol (PAR) [...] Read more.
A novel liquid chromatography-tandem mass spectrometry (LC-MS/MS) method has been developed and validated for the simultaneous determination of tipiracil (TIP), trifluridine (FTD), and their metabolites, 5-trifluoromethyluracil (FTY) and 5-carboxy-2′-deoxyuridine (5CDU), in rat plasma. This method is highly sensitive, specific, and fast. Paracetamol (PAR) is used as an internal standard (IS). Using acetonitrile-induced protein precipitation, the analytes were extracted from a plasma sample and separated on a Waters BEH C18 (1.7 μm particle size, 50 mm × 2.1 mm ID) column protected by a security guard cartridge (C18, 4 × 2.0 mm). The isocratic mobile phase was made up of methanol and water containing 0.1% formic acid (80:20, v/v) at a flow rate of 0.5 mL/min for 4 min. The quantification was performed using a positive electrospray ionization (ESI) interface and a multiple-reaction monitoring (MRM) mode. The MRM transitions employed were m/z 242.96 → 182.88 for TIP, 296.96 → 116.86 for FTD, 180.98 → 139.85 for FTY, 272.96 → 156.86 for 5CDU, and 151.97 → 92.68 for IS. The validated method complied with the guidelines set by the US-FDA over on a linear concentration range of 5–4000 ng/mL for FTD, FTY, and 5CDU, and 5–1000 ng/mL for TIP. The coefficient of determination (r2) was equal to or greater than 0.997. The corresponding lower limits of detection (LLOD) were 1.5 ng/mL for FTD, FTY, and 5CDU and 1.0 ng/mL for TIP. The recoveries of all analytes from rat plasma ranged from 88.67% to 112.18%, and the mean relative standard deviation (RSD) of accuracy and precision result was less than or equal to 6.84%. FTD, FTY, 5CDU, and TIP demonstrated adequate stability throughout the various circumstances examined. Additionally, no matrix effects were identified for any of the analytes. The assay was effectively utilized to conduct a pharmacokinetic study in rats following the oral administration of FTD and TIP at a dosage of 5.6 mg/kg, with a ratio of 1:0.5 for FTD and TIP, respectively. This indicates that the suggested approach is suitable for future clinical research. The pharmacokinetic parameters Cmax (maximum concentration), Tmax (time to reach maximum concentration), t1/2 (half-life), AUC0-24 (area under the concentration–time curve from 0 to 24 h), AUC total (total area under the concentration–time curve), Ke (elimination rate constant), Vd (volume of distribution), and CL (clearance) of all analytes were assessed. The assay developed exhibits significant advancements compared to earlier bioanalytical methods documented in the literature. These improvements include high sensitivity, specificity, and efficacy in high throughput analysis of complex matrices. Additionally, the assay offers a shorter run time and smaller sample volume (50 μL). Full article
(This article belongs to the Special Issue Application of Hyphenated Techniques in Bioanalysis and Metabolite Identification)
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24 pages, 4875 KiB  
Article
Biosynthesis of Salbutamol-4′-O-sulfate as Reference for Identification of Intake Routes and Enantiopure Salbutamol Administration by Achiral UHPLC-MS/MS
by Annika Lisa Jendretzki, Lukas Corbinian Harps, Yanan Sun, Felix Bredendiek, Matthias Bureik, Ulrich Girreser, Xavier de la Torre, Francesco M. Botrè and Maria Kristina Parr
Separations 2023, 10(8), 427; https://doi.org/10.3390/separations10080427 - 28 Jul 2023
Cited by 1 | Viewed by 2327
Abstract
The aim of the study was a comprehensive and quantitative determination of salbutamol and its sulfoconjugated major metabolite in urine samples using achiral ultrahigh performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS). Therefore, salbutamol-4′-O-sulfate was biosynthesized as a reference using genetically modified fission [...] Read more.
The aim of the study was a comprehensive and quantitative determination of salbutamol and its sulfoconjugated major metabolite in urine samples using achiral ultrahigh performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS). Therefore, salbutamol-4′-O-sulfate was biosynthesized as a reference using genetically modified fission yeast cells, and the product was subsequently characterized by NMR and HRMS. In competitive sports, salbutamol is classified as a prohibited drug; however, inhalation at therapeutic doses is permitted with a maximum allowance of 600 µg/8 h. In contrast, the enantiopure levosalbutamol is prohibited under any condition. For analytical discrimination, the amount of salbutamol and its main metabolite excreted in the urine was studied. As proof of concept, a longitudinal study in one healthy volunteer was performed in order to investigate excreted amounts and to study potential discrimination using achiral chromatography. Discrimination of administration of racemic salbutamol or the enantiopure levosalbutamol was not achieved by solely analyzing salbutamol as the parent compound. However, a distinction was possible by evaluation of the proportion of salbutamol-4′-O-sulfate in relation to salbutamol. Therefore, reference material of metabolites is of great importance in doping control, especially for threshold substances. Full article
(This article belongs to the Special Issue Application of Hyphenated Techniques in Bioanalysis and Metabolite Identification)
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23 pages, 2691 KiB  
Article
Quantitation of Formoterol, Salbutamol, and Salbutamol-4′-O-Sulfate in Human Urine and Serum via UHPLC-MS/MS
by Lukas C. Harps, Daniel A. Bizjak, Ulrich Girreser, Martina Zügel, Jürgen M. Steinacker, Patrick Diel and Maria Kristina Parr
Separations 2023, 10(7), 368; https://doi.org/10.3390/separations10070368 - 22 Jun 2023
Cited by 4 | Viewed by 1654
Abstract
The adrenergic beta-2 agonists formoterol and salbutamol are used for the treatment of asthma and COPD but are also misused in sports competitions. Therefore, they are included in WADA regulations. Both drugs are mainly excreted in urine after administration via inhalation. A four-armed, [...] Read more.
The adrenergic beta-2 agonists formoterol and salbutamol are used for the treatment of asthma and COPD but are also misused in sports competitions. Therefore, they are included in WADA regulations. Both drugs are mainly excreted in urine after administration via inhalation. A four-armed, double-blind cross-over clinical trial was conducted involving endurance-trained participants (12 females and 12 males). Inhalation dosages of 36 μg formoterol, 1200 μg salbutamol, a combination of both, or a placebo were administered before exercise. Serum and urine were collected after exercise and 3 and 24 h after administration. Here, we show the successful quantitation of formoterol, salbutamol, and its phase II metabolite salbutamol-4′-O-sulfate in all urine and serum samples using ultra-high performance liquid chromatography–tandem mass spectrometry. In the serum analysis, results of up to 14.2 pg/mL formoterol, 10.0 ng/mL salbutamol, and 21.4 ng/mL salbutamol-4′-O-sulfate (calculated as salbutamol equivalent) were found. In urine, maximum concentrations (after deglucuronidation) were 17.2 ng/mL formoterol, 948.5 ng/mL salbutamol, and 2738.5 ng/mL salbutamol-4′-O-sulfate. Sex-specific differences in serum concentrations as well as in urinary excretion were observed. The results pronounce the importance of the implementation and elucidation of phase II metabolites to quantitation methods in antidoping. Full article
(This article belongs to the Special Issue Application of Hyphenated Techniques in Bioanalysis and Metabolite Identification)
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18 pages, 1391 KiB  
Article
Chemometric Approach for Profiling of Metabolites of Potential Antioxidant Activity in Apiaceae Species Based on LC-PDA-ESI-MS/MS and FT-NIR
by Noha H. Atta, Heba Handoussa, Iris Klaiber, Bernd Hitzmann and Rasha S. Hanafi
Separations 2023, 10(6), 347; https://doi.org/10.3390/separations10060347 - 8 Jun 2023
Cited by 1 | Viewed by 1601
Abstract
Chemometrics is a tool for data mining and unlocking the door for solving big data queries. Apiaceae is a family species which is commonly cultivated worldwide. Although members of this species are widely used as antioxidant, antibacterial, antifungal, and anti-inflammatory agents, their metabolites [...] Read more.
Chemometrics is a tool for data mining and unlocking the door for solving big data queries. Apiaceae is a family species which is commonly cultivated worldwide. Although members of this species are widely used as antioxidant, antibacterial, antifungal, and anti-inflammatory agents, their metabolites profiling remains ambiguous. Based on WHO support, chemometrics has been used in evaluating the quality and authenticity of the herbal products. The objective of this study is to profile and characterize phenolic metabolites in nine species from Egyptian cultivars and three different species of German cultivars from the Apiaceae family using multivariate analysis after LC-PDA-ESI-MS/MS and near infrared spectroscopy data are generated. Principal component analysis was successfully applied to distinguish between the nine Egyptian cultivars and the three German cultivars, and hierarchical cluster analysis also confirmed this distinctive clustering. Partial least square regression (PLS-R) models showed a relationship between phytochemicals and antioxidant activities. The metabolites responsible for the clustering pattern and variables important for projection (VIP) were identified, being twelve amongst nine Egyptian cultivar samples and thirteen amongst the Egyptian cultivar and the German cultivar comparison. The identified VIPs were also correlated with the antioxidant activity using PLS-R. In conclusion, the study showed novelty in the application of hyphenated analytical techniques and chemometrics that assist in quality control of herbal medicine. Full article
(This article belongs to the Special Issue Application of Hyphenated Techniques in Bioanalysis and Metabolite Identification)
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Review

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20 pages, 432 KiB  
Review
Challenges in the Analytical Preparation of a Biological Matrix in Analyses of Endocrine-Disrupting Bisphenols
by Nataša Milić, Maja Milanović, Jovana Drljača, Jan Sudji and Nataša Milošević
Separations 2023, 10(4), 226; https://doi.org/10.3390/separations10040226 - 24 Mar 2023
Cited by 5 | Viewed by 2314
Abstract
Endocrine-disrupting chemicals (EDCs) are xenobiotics presented in a variety of everyday products that may disrupt the normal activity of hormones. Exposure to bisphenol A as EDC at trace and ultra-trace levels is associated with adverse health effects, and children are recognized as the [...] Read more.
Endocrine-disrupting chemicals (EDCs) are xenobiotics presented in a variety of everyday products that may disrupt the normal activity of hormones. Exposure to bisphenol A as EDC at trace and ultra-trace levels is associated with adverse health effects, and children are recognized as the most vulnerable group to EDCs exposure. In this review, a summary is presented of up-to-date sample preparation methods and instrumental techniques applied for the detection and quantification of bisphenol A and its structural analogues in various biological matrices. Biological matrices such as blood, cell-free blood products, urine, saliva, breast milk, cordial blood, amniotic and semen fluids, as well as sweat and hair, are very complex; therefore, the detection and later quantification of bisphenols at low levels present a real analytical challenge. The most popular analytical approaches include gas and liquid chromatography coupled with mass spectrometry, and their enhanced reliability and sensitivity finally allow the separation and detection of bisphenols in biological samples, even as ultra-traces. Liquid/liquid extraction (LLE) and solid-phase extraction (SPE) are still the most common methods for their extraction from biological matrices. However, many modern and environmentally safe microextraction techniques are currently under development. The complexity of biological matrices and low concentrations of analytes are the main issues for the limited identification, as well as understanding the adverse health effects caused by chronical and ubiquitous exposure to bisphenols and its analogues. Full article
(This article belongs to the Special Issue Application of Hyphenated Techniques in Bioanalysis and Metabolite Identification)
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