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A special issue of Molecules (ISSN 1420-3049). This special issue belongs to the section "Analytical Chemistry".

Deadline for manuscript submissions: closed (30 April 2022) | Viewed by 20399

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Special Issue Editors

Prof. Dr. Manuel Sergi

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Guest Editor

Faculty of Bioscience and Technology for Food, Agriculture and Environment, University of Teramo, 64100 Teramo, Italy
Interests: liquid chromatography–mass spectrometry; high-resolution mass spectrometry; sample preparation; method validation; forensic toxicology; food contaminants; bioactive compounds

Dr. Camilla Montesano

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Guest Editor

Department of Chemistry, Sapienza University of Rome, 00185 Rome, Italy
Interests: liquid chromatography–mass spectrometry; high-resolution mass spectrometry; sample preparation; forensic toxicology; drug metabolism and pharmacokinetics; metabolomics

Special Issue Information

Dear Colleagues,

The importance of the quantification and/or characterization of drugs as well as endogenous metabolites, proteins, oligonucleotides, etc., is crucial in bioanalytical and biomedical applications. The developments and advances in LC–MS have revolutionized the analytical study of biomolecules by enabling their accurate identification and quantification in an unprecedented manner. We are also witnessing an enormous growth of new technologies, allowing more reliable, faster, and more comprehensive studies than ever before. The typical bioanalytical applications of LC-MS range from therapeutic drug monitoring and pharmacokinetic studies to -omics applications, especially metabolomics and proteomics; in fact, the determination of abnormal proteins or altered metabolic patterns in patients has certainly extended the use of LC-MS in bioanalysis.

This Special Issue aims to provide a forum for the dissemination of the latest information on new approaches, methods and innovative sample preparation techniques in bioanalytical and biomedical applications of LC-MS.

Prof. Dr. Manuel Sergi
Dr. Camilla Montesano
Guest Editors

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Keywords

LC-MS/MS
Drug analysis
Biomarkers
Metabolomics
Biomedical applications
Proteomics

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Published Papers (7 papers)

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Research

14 pages, 1088 KiB

Open AccessArticle

Dystrophin Protein Quantification as a Duchenne Muscular Dystrophy Diagnostic Biomarker in Dried Blood Spots Using Multiple Reaction Monitoring Tandem Mass Spectrometry: A Preliminary Study

by Refat M. Nimer, Khalid M. Sumaily, Arwa Almuslat, Mai Abdel Jabar, Essa M. Sabi, Mohammad A. Al-Muhaizea and Anas M. Abdel Rahman

Molecules 2022, 27(12), 3662; https://doi.org/10.3390/molecules27123662 - 7 Jun 2022

Cited by 5 | Viewed by 2602

Abstract

Duchenne muscular dystrophy (DMD) is an X-linked recessive disorder characterized by progressive muscle loss, leading to difficulties in movement. Mutations in the DMD gene that code for the protein dystrophin are responsible for the development of DMD disorder, where the synthesis of this protein is completely halted. Therefore, circulating dystrophin protein could be a promising biomarker of DMD disease. Current methods for diagnosing DMD have sensitivity, specificity, and reproducibility limitations. Herein, a quantitative liquid chromatography–tandem spectrometry (LC–MS/MS) technique in multiple reaction monitoring (MRM) mode was designed and validated for accurate dystrophin protein measurement in a dried blood spot (DBS). The method was successfully validated on the basis of international guidelines regarding calibration curves, precision, and accuracy. In addition, patients and healthy controls were used to test the amount of dystrophin protein circulating in DBS samples as a potential biomarker for DMD disorders. DMD patients were found to have considerably lower levels than controls. To the best of our knowledge, this is the first study to report dystrophin levels in DBS through LC–MS/MS as a diagnostic marker for DMD to the proposed MRM method, providing a highly specific and sensitive approach to dystrophin quantification in a DBS that can be applied in DMD screening. Full article

(This article belongs to the Special Issue LC-MS in Bioanalysis)

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16 pages, 3781 KiB

Open AccessArticle

Quantitative Analysis of Daporinad (FK866) and Its In Vitro and In Vivo Metabolite Identification Using Liquid Chromatography-Quadrupole-Time-of-Flight Mass Spectrometry

by Minjae Park, Byeong Ill Lee, Jangmi Choi, Yuri Park, Seo-Jin Park, Jeong-Hyeon Lim, Jiyu Lee and Young G. Shin

Molecules 2022, 27(6), 2011; https://doi.org/10.3390/molecules27062011 - 21 Mar 2022

Cited by 1 | Viewed by 2636

Abstract

Daporinad (FK866) is one of the highly specific inhibitors of nicotinamide phosphoribosyl transferase (NAMPT) and known to have its unique mechanism of action that induces the tumor cell apoptosis. In this study, a simple and sensitive liquid chromatography–quadrupole-time-of-flight–mass spectrometric (LC-qTOF-MS) assay has been developed for the evaluation of drug metabolism and pharmacokinetics (DMPK) properties of Daporinad in mice. A simple protein precipitation method using acetonitrile (ACN) was used for the sample preparation and the pre-treated samples were separated by a C18 column. The calibration curve was evaluated in the range of 1.02~2220 ng/mL and the quadratic regression (weighted 1/concentration²) was used for the best fit of the curve with a correlation coefficient ≥ 0.99. The qualification run met the acceptance criteria of ±25% accuracy and precision values for QC samples. The dilution integrity was verified for 5, 10 and 30-fold dilution and the accuracy and precision of the dilution QC samples were also satisfactory within ±25% of the nominal values. The stability results indicated that Daporinad was stable for the following conditions: short-term (4 h), long-term (2 weeks), freeze/thaw (three cycles). This qualified method was successfully applied to intravenous (IV) pharmacokinetic (PK) studies of Daporinad in mice at doses of 5, 10 and 30 mg/kg. As a result, it showed a linear PK tendency in the dose range from 5 to 10 mg/kg, but a non-linear PK tendency in the dose of 30 mg/kg. In addition, in vitro and in vivo metabolite identification (Met ID) studies were conducted to understand the PK properties of Daporinad and the results showed that a total of 25 metabolites were identified as ten different types of metabolism in our experimental conditions. In conclusion, the LC-qTOF-MS assay was successfully developed for the quantification of Daporinad in mouse plasma as well as for its in vitro and in vivo metabolite identification. Full article

(This article belongs to the Special Issue LC-MS in Bioanalysis)

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11 pages, 5130 KiB

Open AccessArticle

LC-MS/MS Analysis Elucidates a Daily Rhythm in Orexin A Concentration in the Rat Vitreous Body

by Lukasz Chrobok, Sylwia Bajkacz, Jasmin Daniela Klich and Marian Henryk Lewandowski

Molecules 2021, 26(16), 5036; https://doi.org/10.3390/molecules26165036 - 19 Aug 2021

Cited by 6 | Viewed by 2272

Abstract

Orexins are two neuropeptides synthesised mainly in the brain lateral hypothalamic area. The orexinergic system provides arousal-dependent cues for a plethora of brain centres, playing a vital role in feeding behaviour, regulation of the sleep–wake cycle and circadian rhythms. Recently, orexins were found to be produced in the retina of an eye; however, their content in the vitreous body and possible daily pattern of expression have not yet been explored. In this manuscript, we describe the development and validation of a liquid chromatography with tandem mass spectrometry (LC-MS/MS) method designed for quantitative bioanalysis of orexin in the rat vitreous body. Orexin was extracted from vitreous body samples with a water:acetonitrile:formic acid (80:20:0.1; v/v/v) mixture followed by vortexing and centrifuging. Separation was performed on a reverse-phase HPLC column under gradient conditions. Orexin was analysed via multiple-reaction monitoring (MRM) in the positive electrospray mode. The total analysis time for each sample was less than 5.0 min. Once the method was fully optimised, it was then validated, following the 2018 FDA guidance on bioanalytical method validations. The calibration curves for orexin (1–500 ng/mL) were constructed using a linear regression with a 1/x² weighting. The lower limit of quantitation for orexin was 1.0 pg/mL for the vitreous body. Intra-day and inter-day estimates of accuracy and precision were within 10% of their nominal values, indicating that the method is reliable for quantitation of orexin in the rat vitreous body. From the physiological perspective, our results are the first to show daily rhythm of orexin synthesis by the retina with possible implications on the circadian regulation of vision. Full article

(This article belongs to the Special Issue LC-MS in Bioanalysis)

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14 pages, 1251 KiB

Open AccessArticle

Eco-Friendly UPLC–MS/MS Method for Determination of a Fostamatinib Metabolite, Tamatinib, in Plasma: Pharmacokinetic Application in Rats

by Essam Ezzeldin, Muzaffar Iqbal, Yousif A. Asiri, Ahmed Y. A. Sayed and Rashad Alsalahi

Molecules 2021, 26(15), 4663; https://doi.org/10.3390/molecules26154663 - 31 Jul 2021

Cited by 2 | Viewed by 2716

Abstract

Fostamatinib is a prodrug of the active metabolite tamatinib, which is a spleen tyrosine kinase (Syk) inhibitor used in the treatment of primary chronic adult immune thrombocytopenia and rheumatoid arthritis. A highly sensitive, rapid, reliable, and green method was developed and validated using ultra-performance liquid chromatography and tandem mass spectrometry (UPLC–MS/MS) for quantification of tamatinib in rat plasma. Ibrutinib was used as internal standard and liquid–liquid extraction was applied using tert-butyl methyl ether. The analyte was separated on an Acquity^TM CSH C₁₈ (2.1 mm × 100 mm, 1.7 µm) column using mobile phase consisting of 10 mM ammonium acetate and acetonitrile (10:90) and the flow rate was 0.25 mL/min. Electrospray ionization (ESI) was carried out in positive mode. Quantitation of tamatinib and the IS was performed using multiple reaction monitoring mode with precursor-to-product transitions of m/z 471.1 > 122.0 and m/z 441.1 > 84.0, respectively. The calibration range was 0.1–1000.0 ng/mL and the linearity of the method was ≥0.997. The developed method greenness was investigated. All principal parameters for the method, including linearity, accuracy, precision, recovery, and stability, were within acceptable ranges. Tamatinib pharmacokinetic study in rats was successfully carried out using the developed method. Full article

(This article belongs to the Special Issue LC-MS in Bioanalysis)

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16 pages, 5720 KiB

Open AccessArticle

Development and Validation of Liquid Chromatography-Tandem Mass Spectrometry Methods for the Quantification of Cefquinome, Ceftiofur, and Desfuroylceftiofuracetamide in Porcine Feces with Emphasis on Analyte Stability

by Sofie Rutjens, Siska Croubels, Siegrid De Baere and Mathias Devreese

Molecules 2021, 26(15), 4598; https://doi.org/10.3390/molecules26154598 - 29 Jul 2021

Cited by 2 | Viewed by 2319

Abstract

Cefquinome and ceftiofur are β-lactam antibiotics used for the treatment of bacterial infections in swine. Although these antimicrobials are administered intramuscularly, the exposure of the gut microbiota to these cephalosporins is not well described. This exposure can contribute to the emergence and spread of antimicrobials in the environment and to the possible spread of antimicrobial resistance genes. To assess the impact of drug administration on the intestinal excretion of these antimicrobials it is essential to measure the amounts of native compound and metabolites in feces. Two (ultra)-high-performance liquid chromatography-tandem mass spectrometry ((U)HPLC–MS/MS) methods were developed and validated, one for the determination of cefquinome and ceftiofur and the other for the determination of ceftiofur residues, measured as desfuroylceftiofuracetamide, in porcine feces. The matrix-based calibration curve was linear from 5 ng g⁻¹ to 1000 ng g⁻¹ for cefquinome (correlation coefficient (r) = 0.9990 ± 0.0007; goodness of fit (gof) = 3.70 ± 1.43) and ceftiofur (r = 0.9979 ± 0.0009; gof = 5.51 ± 1.14) and quadratic from 30 ng g⁻¹ to 2000 ng g⁻¹ for desfuroylceftiofuracetamide (r = 0.9960 ± 0.0020; gof = 7.31 ± 1.76). The within-day and between-day precision and accuracy fell within the specified ranges. Since β-lactam antibiotics are known to be unstable in feces, additional experiments were conducted to adjust the sampling protocol in order to minimize the impact of the matrix constituents on the stability of the analytes. Immediately after sampling, 500 µL of an 8 µg mL⁻¹ tazobactam solution in water was added to 0.5 g feces, to reduce the degradation in matrix. Full article

(This article belongs to the Special Issue LC-MS in Bioanalysis)

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15 pages, 1448 KiB

Open AccessArticle

Characterization of Metabolites in Plasma, Urine and Feces of Healthy Participants after Taking Brahmi Essence for Twelve Weeks Using LC-ESI-QTOF-MS Metabolomic Approach

by Genet Minale, Tongchai Saesong, Prapapan Temkitthawon, Neti Waranuch, Nitra Nuengchamnong, Krongkarn Chootip, Natakorn Kamkaew, Teeraporn Kongbangkerd, Jinutda Engsuwan and Kornkanok Ingkaninan

Molecules 2021, 26(10), 2944; https://doi.org/10.3390/molecules26102944 - 15 May 2021

Cited by 7 | Viewed by 3568

Abstract

Brahmi essence, developed from Bacopa monnieri (L.) Wettst. standardized extract and mulberry juice, was proven to improve the memory speed of healthy participants aged 55–80 years old, following a 12-week dietary program. However, the metabolites have not yet been reported. Our objective was to characterize the altered metabolites in the plasma, urine, and feces of healthy volunteers after consumption of Brahmi essence for 12 weeks, using the LC-MS metabolomics approach. The altered metabolites were selected from OPLS-DA S-plots; 15 metabolites in the plasma, 7 in the urine, and 17 in the feces samples were tentatively identified by comparison with an online database and literature. The metabolites in the plasma samples were in the classes of amino acids, acylcarnitine, and phospholipids. Benzeneactamide-4-O-sulphate and 3-hydroxyhippuric acid were found in urine samples. The metabolites in the class of amino acids, together with jujubogenin and pseudojujubogenin, were identified in the fecal samples. The aminoacyl-tRNA, aromatic amino acids, and branched-chain amino acid biosynthetic pathways were mainly related to the identified metabolites in all three samples. It could be implied that those metabolites and their pathways might be linked with the effect of Brahmi essence on memory speed. Full article

(This article belongs to the Special Issue LC-MS in Bioanalysis)

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12 pages, 1151 KiB

Open AccessArticle

Serial Hydrolysis for the Simultaneous Analysis of Catecholamines and Steroids in the Urine of Patients with Alopecia Areata

by Yu-Ra Lee, Bark-Lynn Lew, Woo-Young Sim, Jongki Hong and Bong-Chul Chung

Molecules 2021, 26(9), 2734; https://doi.org/10.3390/molecules26092734 - 6 May 2021

Cited by 3 | Viewed by 2328

Abstract

Catecholamines and steroids are well-known neurotransmitters and hormones that rapidly change the excitability of neurons. Alopecia areata is a disease for which the exact cause is unknown, but it is considered to be associated with stress, and so the simultaneous analysis of catecholamines and steroids is required for the diagnosis of alopecia areata. Thus, we herein report the simultaneous analysis of catecholamines and steroids bearing different functional groups for the first time, during which it was necessary to carry out a serial hydrolysis procedure. Following hydrolysis of the urine samples to produce the free forms from the urinary conjugates, ethyl acetate extractions were carried out, and chemical derivatization was performed using dansyl chloride to increase the sensitivity of the liquid chromatography–tandem mass spectrometry method. The matrix effects and recoveries of this analytical method were validated, giving values of 85.4–122.9% and 88.8–123.0%, respectively. In addition, the method accuracy and precision were assessed, giving values of 0.4–21.5% and 2.0–21.6% for the intra-day and inter-day precisions, respectively. This validated method was then applied to identify differences between patients with and without alopecia areata, wherein the metanephrine content was found to be significantly higher in the alopecia areata patient group. This quantitative profiling method can also be applied to steroid-dependent diseases, as well as catecholamine-related diseases. Full article

(This article belongs to the Special Issue LC-MS in Bioanalysis)

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