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

Deadline for manuscript submissions: closed (1 November 2021) | Viewed by 32929

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Prof. Dr. James Barker

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Department of Chemical and Pharmaceutical Sciences, School of Life Sciences, Pharmacy and Chemistry, Kingston University, Kingston upon Thames, London KT1 2EE, UK
Interests: pharmaceutical and forensic analysis; environmental analytical chemistry; drugs of abuse; drug/drug interactions; method development for enviro/bioanalysis; separation methods (GC, HPLC, LC–MS/MS); mass spectrometry and isotope ratio mass spectrometry; development and application of innovative sorbents for extraction of pollutants from water; fate and behaviour of organic contaminants, drugs, toxic metals, pesticides and emerging pollutants in the environment and during wastewater treatment; analysis of bioactive species from herbal matrices, application of nuclear instrumental methods for analysis
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Dr. Iltaf Shah

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Department of Chemistry, College of Science, UAE University, Al Ain, UAE
Interests: clinical biochemistry; bioanalytical chemistry; forensic biochemistry and nutrition; drug delivery; development; validation; and analysis; drugs of abuse; steroids and antioxidants’ bioanalysis in human body matrices (especially hair); nutrition; toxicology; spectroscopy; separation science; chromatography; and mass spectrometry; vitamins; (especially vitamin D) bioanalyses as markers for different diseases

Special Issue Information

Dear Colleagues,

Mass spectrometry involves the measurement of the mass-to-charge ratio of ions. It has become an essential analytical tool in biological research and can characterize a wide variety of biomolecules, such as carbohydrates, proteins, and nucleotides. The versatility of mass spectrometry has allowed it to become a vital tool in biological research. Submitted articles for this issue should be related to qualitative and quantitative information on the elemental, isotopic, and molecular constituents of samples; samples from the gas, liquid, or solid phases, and masses that can be studied in the range from single atoms to proteins (over 300,000 Da).

The submitted articles could belong to a wide variety of different areas: e.g., the biological applications of MS for screening newborns for metabolic disorders, comparing protein expression levels between cells grown in different media, determining the bioavailability of minerals in food, studying how pharmaceutical drugs are metabolized in vivo, soft desorption ionization methods for mass spectrometric analysis of biological macromolecules, the structure of nucleotides and proteins, peptide mass fingerprinting techniques, isotope-coded affinity tags for the possible quantification of individual proteins in complex mixtures, enzymes and coenzymes structure and function determination (including vitamins and cofactors), and pollutant degradation by enzymes in wastewater and forensic applications of MS.

Dr. James Barker
Dr. Iltaf Shah
Guest Editors

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Keywords

mass spectrometry
proteins
enzymes
pharmaceutical drugs
minerals
antioxidants
vitamins
drugs of abuse
toxicology
wastewater treatment

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Published Papers (9 papers)

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Research

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11 pages, 1838 KiB

Open AccessArticle

A Fast and Selective Approach for Profiling Vicinal Diols Using Liquid Chromatography-Post Column Derivatization-Double Precursor Ion Scanning Mass Spectrometry

by Debin Wan, Christophe Morisseau, Bruce D. Hammock and Jun Yang

Molecules 2022, 27(1), 283; https://doi.org/10.3390/molecules27010283 - 3 Jan 2022

Cited by 4 | Viewed by 2279

Abstract

Vicinal diols are important signaling metabolites of various inflammatory diseases, and some of them are potential biomarkers for some diseases. Utilizing the rapid reaction between diol and 6-bromo-3-pyridinylboronic acid (BPBA), a selective and sensitive approach was established to profile these vicinal diols using liquid chromatography-post column derivatization coupled with double precursor ion scan-mass spectrometry (LC-PCD-DPIS-MS). After derivatization, all BPBA-vicinal-diol esters gave a pair of characteristic isotope ions resulting from ⁷⁹Br and ⁸¹Br. The unique isotope pattern generated two characteristic fragment ions of m/z 200 and 202. Compared to a traditional offline derivatization technique, the new LC-PCD-DPIS-MS method retained the capacity of LC separation. In addition, it is more sensitive and selective than a full scan MS method. As an application, an in vitro study of the metabolism of epoxy fatty acids by human soluble epoxide hydrolase was tested. These vicinal-diol metabolites of individual regioisomers from different types of polyunsaturated fatty acids were easily identified. The limit of detection (LOD) reached as low as 25 nM. The newly developed LC-PCD-DPIS-MS method shows significant advantages in improving the selectivity and therefore can be employed as a powerful tool for profiling vicinal-diol compounds from biological matrices. Full article

(This article belongs to the Special Issue Application of LC–MS/MS to Biochemistry)

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10 pages, 322 KiB

Open AccessArticle

A Novel LC–MS/MS-Based Method for the Diagnosis of ADA2 Deficiency from Dried Plasma Spot

by Alessia Cafaro, Federica Pigliasco, Sebastiano Barco, Federica Penco, Francesca Schena, Roberta Caorsi, Stefano Volpi, Gino Tripodi, Marco Gattorno and Giuliana Cangemi

Molecules 2021, 26(18), 5707; https://doi.org/10.3390/molecules26185707 - 21 Sep 2021

Cited by 10 | Viewed by 2399

Abstract

Adenosine Deaminase 2 Deficiency (DADA2) (OMIM: 607575) is a monogenic, autoinflammatory disease caused by the loss of functional homozygous or heterozygous mutations in the ADA 2 gene (previously CECR1, Cat Eye Syndrome Chromosome Region 1). A timely diagnosis is crucial to start Anti-TNF therapies that are efficacious in controlling the disease. The confirmation of DADA2 is based on DNA sequencing and enzymatic assay. It is, thus, very important to have robust and reliable assays that can be rapidly utilized in specialized laboratories that can centralize samples from other centers. In this paper, we show a novel enzymatic assay based on liquid chromatography-tandem mass spectrometry that allows the accurate determination of the ADA2 enzyme activity starting from very small amounts of plasma spotted on filter paper (dried plasma spot). The method allows significantly distinguishing healthy controls from affected patients and carriers and could be of help in implementing the diagnostic workflow of DADA2. Full article

(This article belongs to the Special Issue Application of LC–MS/MS to Biochemistry)

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14 pages, 2178 KiB

Open AccessArticle

An LC-MS/MS Method for Analysis of Vitamin D Metabolites and C3 Epimers in Mice Serum: Oral Supplementation Compared to UV Irradiation

by Amir Sohail, Asma Al Menhali, Soleiman Hisaindee and Iltaf Shah

Molecules 2021, 26(17), 5182; https://doi.org/10.3390/molecules26175182 - 26 Aug 2021

Cited by 7 | Viewed by 3916

Abstract

Introduction: The most common forms of vitamin D in human and mouse serum are vitamin D3 and vitamin D2 and their metabolites. The aim of this study is to determine whether diet and sunlight directly affect the circulating concentrations of vitamin D metabolites in a mouse model. We investigated the serum concentrations of eight vitamin D metabolites—vitamin D (vitamin D3 + vitamin D2), 25OHD (25OHD3 + 25OHD2), 1α25(OH)₂D (1α25(OH)₂D2, and 1α25(OH)₂D3)—including their epimer, 3-epi-25OHD (3-epi-25OHD3 and 3-epi-25OHD2), and a bile acid precursor 7alpha-hydroxy-4-cholesten-3-one (7αC4), which is known to cause interference in liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis. Method: The LC-MS/MS method was validated according to FDA-US guidelines. The validated method was used for the analysis of mouse serum samples. Forty blood samples from mice were collected and divided into three groups. The first group, the DDD mice, were fed a vitamin D-deficient diet (25 IU VD3/kg of diet) and kept in the dark; the second group, the SDD mice, were maintained on a standard-vitamin D diet (1000 IU VD3) and kept in the dark; and the third group, SDL, were fed a standard-vitamin D diet (1000 IU VD3) but kept on a normal light/dark cycle. LC-MS/MS was used for the efficient separation and quantitation of all the analytes. Results: The validated method showed good linearity and specificity. The intraday and interday precision were both <16%, and the accuracy across the assay range was within 100 ± 15%. The recoveries ranged between 75 and 95%. The stability results showed that vitamin D metabolites are not very stable when exposed to continuous freeze–thaw cycles; the variations in concentrations of vitamin D metabolites ranged between 15 and 60%. The overlapping peaks of vitamin D, its epimers, and its isobar (7αC4) were resolved using chromatographic separation. There were significant differences in the concentrations of all metabolites of vitamin D between the DDD and SDL mice. Between the groups SDD (control) and SDL, a significant difference in the concentrations of 3-epi-25OHD was noted, where C3 epimer was about 30% higher in SDL group while no significant differences were noted in the concentrations of vitamin D, 25OHD, 1α25(OH)₂D, and 7αC4 between SDD and SDL group. Conclusions: A validated method, combined with a simple extraction technique, for the sensitive LC-MS/MS determination of vitamin D metabolites is described here. The method can eliminate the interferences in LC-MS/MS analysis caused by the overlapping epimer and isobar due to them having the same molecular weights as 25OHD. The validated method was applied to mouse serum samples. It was concluded that a standard-vitamin D diet causes an increase in the proportion of all the vitamin D metabolites and C3 epimers and isobar, while UV light has no pronounced effect on the concentrations of the majority of the vitamin D metabolites except 3-epi-25OHD. Further studies are required to confirm this observation in humans and to investigate the biochemical pathways related to vitamin D’s metabolites and their epimers. Full article

(This article belongs to the Special Issue Application of LC–MS/MS to Biochemistry)

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11 pages, 2325 KiB

Open AccessArticle

High Performance Liquid Chromatography–Tandem Mass Spectrometry Method for Correlating the Metabolic Changes of Lactate, Pyruvate and L-Glutamine with Induced Tamoxifen Resistant MCF-7 Cell Line Potential Molecular Changes

by Ala A. Alhusban, Sokiyna Albustanji, Lama A. Hamadneh and Aliaa I. Shallan

Molecules 2021, 26(16), 4824; https://doi.org/10.3390/molecules26164824 - 10 Aug 2021

Cited by 9 | Viewed by 3884

Abstract

Breast cancer is one of the most prevalent cancers worldwide usually treated with Tamoxifen. Tamoxifen resistance development is the most challenging issue in an initially responsive breast tumor, and mechanisms of resistance are still under investigation. The objective of this study is to develop and validate a selective, sensitive, and simultaneous high performance liquid chromatography–tandem mass spectrometry method to explore the changes in substrates and metabolites in supernatant media of developed Tamoxifen resistance MCF-7 cells. We focus on the determination of lactate, pyruvate, and L-glutamine which enables the tracking of changes in metabolic pathways as a result of the resistance process. Chromatographic separation was achieved within 3.5 min. using a HILIC column (4.6 × 100 mm, 3.5 µm particle size) and mobile phase of 0.05 M acetic acid–ammonium acetate buffer solution pH 3.0: Acetonitrile (40:60 v/v). The linear range was 0.11–2.25, 0.012–0.227, and 0.02–0.20 mM for lactate, pyruvate, and L-glutamine, respectively. Within- and between-run accuracy was in the range 98.94-105.50% with precision (CV, %) of ≤0.86%. The results revealed a significant increase in both lactate and pyruvate production after acquiring the resistant. An increase in L-glutamine levels was also observed and could be attributed to its over production or decline in its consumption. Therefore, further tracking of genes responsible of lactate, pyruvate, and glutamine metabolic pathways should be performed in parallel to provide in-depth explanation of resistance mechanism. Full article

(This article belongs to the Special Issue Application of LC–MS/MS to Biochemistry)

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13 pages, 2054 KiB

Open AccessArticle

Simplified and Rapid Determination of Primaquine and 5,6-Orthoquinone Primaquine by UHPLC-MS/MS: Its Application to a Pharmacokinetic Study

by Waritda Pookmanee, Siriwan Thongthip, Jeeranut Tankanitlert, Mathirut Mungthin, Chonlaphat Sukasem and Supeecha Wittayalertpanya

Molecules 2021, 26(14), 4357; https://doi.org/10.3390/molecules26144357 - 19 Jul 2021

Cited by 5 | Viewed by 2361

Abstract

The method for the determination of primaquine (PQ) and 5,6-orthoquinone primaquine (5,6-PQ), the representative marker for PQ active metabolites, via CYP2D6 in human plasma and urine has been validated. All samples were extracted using acetonitrile for protein precipitation and analyzed using the ultra-high-performance liquid chromatography–tandem mass spectrometry (UHPLC-MS/MS) system. Chromatography separation was carried out using a Hypersil GOLD^TM aQ C₁₈ column (100 × 2.1 mm, particle size 1.9 μm) with a C₁₈ guard column (4 × 3 mm) flowed with an isocratic mode of methanol, water, and acetonitrile in an optimal ratio at 0.4 mL/min. The retention times of 5,6-PQ and PQ in plasma and urine were 0.8 and 1.6 min, respectively. The method was validated according to the guideline. The linearity of the analytes was in the range of 25–1500 ng/mL. The matrix effect of PQ and 5,6-PQ ranged from 100% to 116% and from 87% to 104% for plasma, and from 87% to 89% and from 86% to 87% for urine, respectively. The recovery of PQ and 5,6-PQ ranged from 78% to 95% and form 80% to 98% for plasma, and from 102% to from 112% to 97% to 109% for urine, respectively. The accuracy and precision of PQ and 5,6-PQ in plasma and urine were within the acceptance criteria. The samples should be kept in the freezer (−80 °C) and analyzed within 7 days due to the metabolite stability. This validated UHPLC-MS/MS method was beneficial for a pharmacokinetic study in subjects receiving PQ. Full article

(This article belongs to the Special Issue Application of LC–MS/MS to Biochemistry)

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10 pages, 1128 KiB

Open AccessArticle

Determination of the Main Ergot Alkaloids and Their Epimers in Oat-Based Functional Foods by Ultra-High Performance Liquid Chromatography Tandem Mass Spectrometry

by Laura Carbonell-Rozas, Laura Gámiz-Gracia, Francisco J. Lara and Ana M. García-Campaña

Molecules 2021, 26(12), 3717; https://doi.org/10.3390/molecules26123717 - 18 Jun 2021

Cited by 8 | Viewed by 2667

Abstract

An ultra-high performance liquid chromatography coupled to tandem mass spectrometry method is proposed for the determination of the major ergot alkaloids (ergometrine, ergosine, ergotamine, ergocornine, ergokryptine, ergocristine) and their epimers (ergometrinine, ergosinine, ergotaminine, ergocorninine, ergokryptinine, and ergocristinine) in oat-based foods and food supplements. A modified QuEChERS (quick, easy, cheap, effective, rugged, and safe) procedure was applied as sample treatment, reducing the consumption of organic solvent and increasing sensitivity. This method involved an extraction with acetonitrile and ammonium carbonate (85:15, v/v) and a clean-up step based on dispersive solid-phase extraction, employing a mixture of C18/Z-Sep+ as sorbents. Procedural calibration curves were established and limits of quantification were below 3.2 μg/kg for the studied compounds. Repeatability and intermediate precision (expressed as RSD%) were lower than 6.3% and 15%, respectively, with recoveries ranging between 89.7% and 109%. The method was applied to oat-based products (bran, flakes, flour, grass, hydroalcoholic extracts, juices, and tablets), finding a positive sample of oat bran contaminated with ergometrine, ergosine, ergometrinine, and ergosinine (total content of 10.7 μg/kg). Full article

(This article belongs to the Special Issue Application of LC–MS/MS to Biochemistry)

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10 pages, 792 KiB

Open AccessArticle

Application of an LC–MS/MS Method for the Simultaneous Quantification of Homovanillic Acid and Vanillylmandelic Acid for the Diagnosis and Follow-Up of Neuroblastoma in 357 Patients

by Narae Hwang, Eunbin Chong, Hyeonju Oh, Hee Won Cho, Ji Won Lee, Ki Woong Sung and Soo-Youn Lee

Molecules 2021, 26(11), 3470; https://doi.org/10.3390/molecules26113470 - 7 Jun 2021

Cited by 15 | Viewed by 3570

Abstract

Homovanillic acid (HVA) and vanillylmandelic acid (VMA) are end-stage metabolites of catecholamine and are clinical biomarkers for the diagnosis of neuroblastoma. For the first time in Korea, we implemented and validated a liquid chromatography tandem mass spectrometry (LC–MS/MS) assay to measure urinary concentrations of HVA and VMA according to Clinical and Laboratory Standards Institute guidelines. Our LC–MS/MS assay with minimal sample preparation was validated for linearity, lower limit of detection (LOD), lower limit of quantification (LLOQ), precision, accuracy, extraction recovery, carryover, matrix effect, and method comparison. A total of 1209 measurements was performed to measure HVA and VMA in spot urine between October 2019 and September 2020. The relationship between the two urinary markers, HVA and VMA, was analyzed and exhibited high agreement (89.1% agreement, kappa’s k = 0.6) and a strong correlation (Pearson’s r = 0.73). To our knowledge, this is the first study to utilize LC–MS/MS for simultaneous quantitation of spot urinary HVA and VMA and analyze the clinical application of both markers on a large scale for neuroblastoma patients. Full article

(This article belongs to the Special Issue Application of LC–MS/MS to Biochemistry)

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10 pages, 6090 KiB

Open AccessArticle

A Non-Invasive Hair Test to Determine Vitamin D₃ Levels

by Iltaf Shah, Mohammad Mansour, Sheikh Jobe, Emadaldeen Salih, Declan Naughton and Syed Salman Ashraf

Molecules 2021, 26(11), 3269; https://doi.org/10.3390/molecules26113269 - 28 May 2021

Cited by 4 | Viewed by 2922

Abstract

Vitamin D deficiency is being recognized as a global issue and has been implicated in many health issues. Hence, there is an increased interest in developing sensitive, reproducible, and non-invasive assays to measure Vitamin D levels. This study aimed to apply a sensitive liquid chromatography-mass spectrometric assay to hair samples to develop and validate a clinical assay to provide a quarterly average level of vitamin D in one test. Hair samples were collected from 70 male university students/young adults and pulverized/sonicated in methanol/water for 2 h to extract Vitamin D metabolites. A sensitive liquid chromatographic-mass spectrometric assay was employed to quantitate vitamin D and metabolites. Of the eight Vitamin D and metabolites screened, only the primary, clinically significant form of vitamin D (25OHD₃) was detected and quantified in hair samples in the range of 17–1541 pg/mg. One-third of the hair samples (21 out of 70) had Vitamin D levels below the LLOD of the assay (10 pg/mg). The mean and standard deviation values for hair (25OHD₃) were 276.7 ± 329.9, respectively. This pilot study reveals the potential of the vitamin D hair test in clinical assays as a complementary test to a vitamin D blood test, which would provide a quarterly average. Full article

(This article belongs to the Special Issue Application of LC–MS/MS to Biochemistry)

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Review

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28 pages, 5543 KiB

Open AccessReview

Roadside Drug Testing Approaches

by Manal A. Alhefeiti, James Barker and Iltaf Shah

Molecules 2021, 26(11), 3291; https://doi.org/10.3390/molecules26113291 - 29 May 2021

Cited by 15 | Viewed by 7133

Abstract

The purpose of this review is to present an overview of roadside drug testing, driving enforcement, and drunk/drug driving detection around the world. Drunk and drug driving is a severe problem, not only in the UAE, but also around the world. This has important implications for road safety as drunk or drug driving may increase the chances of a driver’s involvement in a road crash when compared to a drug-free driver. Recently, due to increases in drug-impaired drivers’ crash involvement, many mobile roadside drug testing devices have been introduced to the market. These devices use oral fluid, urine or blood matrices. These are on-the-spot tests, which are easy to use and are applied by law enforcement agencies and the public. Law enforcement agencies most commonly use oral fluid to detect the presence of illicit drugs in drivers. This review discusses all the available devices in the market used by the authorities. It also describes the type of drugs widely abused by drivers along with behavioral testing methods. The different types of matrices used for roadside drug testing are also evaluated. Sample collection, storage, and pre-treatment methods are discussed, followed by the confirmatory analysis of positive samples. This article will significantly help law enforcement agencies compare and evaluate all the reliable roadside testing devices and new emerging confirmatory devices available to them in the market. This will help them make an informed decision on which device to adapt to their individual needs. Full article

(This article belongs to the Special Issue Application of LC–MS/MS to Biochemistry)

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