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Chromatographic Analysis of Biological Samples

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A special issue of Separations (ISSN 2297-8739). This special issue belongs to the section "Bioanalysis/Clinical Analysis".

Deadline for manuscript submissions: closed (27 October 2022) | Viewed by 26250

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Prof. Dr. Slawomir Neffe

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Faculty of Advanced Technologies and Chemistry, Military University of Technology, Kaliskiego Street 2, 00-908 Warsaw, Poland
Interests: sampling, processing, and instrumental analysis of environmental samples; chemical warfare agents and hazardous materials; Chemical Weapons Convention

Prof. Dr. Zygfryd Witkiewicz

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Military University of Technology, Faculty of Advanced Technologies and Chemistry, Warsaw, Poland
Interests: chromatographic techniques in the analysis of environmental and biological samples; chemical warfare agents; their precursors and degradation products; separation techniques; sampling and samples processing

Dr. Eduardo Sommella

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Department of Pharmacy, University of Salerno, Fisciano, SA, Italy
Interests: liquid chromatography; peptides; metabolites; high-resolution mass spectrometry
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Special Issue Information

Dear Colleagues,

In recent years, there has been a rapidly growing demand for the analysis of biological materials and samples. Biomedical samples and materials are particularly important in this respect. For the processing, separation, and concentration of analytes, chromatographic and electromigration techniques are most often used, with particular emphasis on liquid chromatography, gas chromatography, ion chromatography or capillary electrophoresis. The identification of very low-level concentrations of analytes in biological samples is often critical for the correct diagnosis of patients and the programming of the correct treatment.

This Special Issue invites contributions on the current advances in and application of chromatographic separation and analytical techniques used for identification and quantification of critical analytes in biological samples.

Prof. Dr. Slawomir Neffe
Prof. Dr. Zygfryd Witkiewicz
Dr. Eduardo Sommella
Guest Editors

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Keywords

sampling and processing of biological material
techniques of separation and concentration of the analytes
chromatographic analysis of biomedical samples
liquid chromatography
gas chromatography
capillary electrophoresis

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Published Papers (10 papers)

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Research

12 pages, 1235 KiB

Open AccessArticle

Liquid Chromatography Fingerprint Analysis of Released Compounds in Plasma Samples of Stroke Patients after Thrombolytic Treatment

by Mar Castellanos, Dolores Fernández-Couto, Andrés Da Silva-Candal, Maria J. Feal-Painceiras, Manuel Rodríguez-Yáñez, Carme Gubern-Mérida and Juan M. Sanchez

Separations 2023, 10(1), 34; https://doi.org/10.3390/separations10010034 - 5 Jan 2023

Cited by 1 | Viewed by 1861

Abstract

Plasma samples obtained from stroke patients treated with recombinant tissue-type plasminogen activator (rt-PA) and not treated with rt-PA were evaluated with different HPLC methodologies to obtain information about the possible release of small molecules as a result of the thrombolytic treatment. Plasma samples, without derivatization and derivatized with 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate (AQC), were evaluated with a HPLC gradient method, which consisted of a mobile phase of 10 mM ammonium acetate buffered solution (pH = 5.3) and acetonitrile. Three different detection methods were applied: UV, fluorescence, and ESI-MS. The results obtained showed that a group of new highly hydrophilic compounds appeared in most samples analyzed from treated patients, just after the administration of rt-PA. These compounds appeared shortly after the administration of the drug and were detected during the first 24 h after treatment, disappearing from plasma after this time. These new compounds were not detected either in controls or in non-treated stroke patients, which suggests that they were released into the plasma as a consequence of the thrombolytic effect of the drug. Our results suggest that these new compounds might be free glycans. The use of AQC as a derivatizing reagent has demonstrated that the new compounds detected cannot contain primary or secondary amine groups in their structure. The molecular mass determined by ESI-MS (821 Da) suggests that if these compounds are free glycans they might be a high-mannose type. Full article

(This article belongs to the Special Issue Chromatographic Analysis of Biological Samples)

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11 pages, 993 KiB

Open AccessArticle

Establishing and Verifying a Robust Liquid Chromatography-Tandem Mass Spectrometry Method to Simultaneously Measure Seven Androgens Present in Plasma Samples

by Songlin Yu, Yutong Zou, Yicong Yin, Jialei Yu, Qianqian Li, Shaowei Xie, Wei Luo, Xiaoli Ma, Danchen Wang and Ling Qiu

Separations 2022, 9(11), 377; https://doi.org/10.3390/separations9110377 - 17 Nov 2022

Cited by 3 | Viewed by 1937

Abstract

Objectives: To develop a robust liquid chromatography-tandem mass spectrometry (LC–MS/MS) method to simultaneously measure seven human plasma androgens, namely testosterone (T), dihydrotestosterone (DHT), androstenedione (A4), dehydroepiandrosterone sulfate (DHEAS), dehydroepiandrosterone (DHEA), 11-ketotestosterone (11-KetoT), and 11β-hydroxytestosterone (11β-OHT). Design and Methods: Plasma was extracted via a solid phase extraction method, and the analytical performance of the assay was validated according to the Clinical & Laboratory Standards Institute guidelines. Overall, 73 apparently healthy volunteers were recruited to evaluate the distribution of these seven androgens; their levels in 25 females with acne and 33 obese females were also evaluated. Results: The developed method exhibited a good precision, with the total coefficient variations (CV) and the intra-assay CVs being within 10%. Furthermore, the recoveries of T, DHT, A4, DHEA, DHEAS, 11-KetoT, and 11β-OHT were 90.3–105.8, 88.7–98.1, 92.4–102.5, 90.5–106.7, 87.6–99.9, 93.3–105.3, and 90.2–104.4%, respectively, and no significant matrix effect was observed after internal standard correction (<20%). Moreover, the limits of quantification were 0.01, 0.01, 0.01, 0.10, 5.00, 0.02, and 0.02 ng/mL for T, DHT, A4, DHEA, DHEAS, 11-KetoT, and 11β-OHT, respectively, which are adequate for their accurate measurement in human plasma samples. It was also determined that patients diagnosed with acne had significantly higher levels of DHT, A4, and DHEAS, while those suffering from obesity had significantly higher levels of T and A4 but lower levels of DHT. Conclusions: A robust LC-MS/MS method for the simultaneous determination of seven androgens in plasma samples was successfully established and validated, which plays important roles in clinical application. Full article

(This article belongs to the Special Issue Chromatographic Analysis of Biological Samples)

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10 pages, 1598 KiB

Open AccessArticle

Sensitive Ion-Chromatographic Determination of Citric Acid in Urine

by Michele Petrarulo, Marta Leporati, Federica Pullara, Maura Frattini, Vita Nannavecchia, Martino Marangella and Domenico Cosseddu

Separations 2022, 9(6), 143; https://doi.org/10.3390/separations9060143 - 6 Jun 2022

Cited by 2 | Viewed by 2679

Abstract

Urine citrate analysis is relevant in the screening and monitoring of patients with calcium nephrolithiasis. A sensitive, fast, easy, and low-maintenance ion chromatographic (IC) method with conductivity detection for the analysis of urine citrate is developed and validated. Its application on true samples is also reported. Sample urine is diluted with a water solution containing internal standard (IS) before the chromatographic assay. The isocratic chromatographic run time is twenty-five minutes, using sodium hydroxide aqueous solution as the mobile phase. The method is fully validated as a quantitative method to objectively demonstrate its applicability for the intended use. The analytical response is linear in the 0.08–10.4 mmol/L concentration range. Precision and accuracy studies carried out on spiked urine and internal quality control samples reveal an imprecision CV% lower than 11% and an accuracy between 85 and 103%. The stability of citrate in urine samples is also evaluated. An easy, rapid, and low-maintenance, cost-effective IC method for urinary citrate determination is developed and validated. Internal standardization improves reliability and precision. The method has been currently used in our laboratory over recent years to analyze more than 1000 samples per year. Full article

(This article belongs to the Special Issue Chromatographic Analysis of Biological Samples)

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15 pages, 2685 KiB

Open AccessEditor’s ChoiceArticle

Measuring Vitamin D₃ Metabolic Status, Comparison between Vitamin D Deficient and Sufficient Individuals

by Laura de los Santos Castillo-Peinado, Mónica Calderón-Santiago, Aura Dulcinea Herrera-Martínez, Soraya León-Idougourram, María Ángeles Gálvez-Moreno, Rafael Luis Sánchez-Cano, Roger Bouillon, Jose Manuel Quesada-Gómez and Feliciano Priego-Capote

Separations 2022, 9(6), 141; https://doi.org/10.3390/separations9060141 - 3 Jun 2022

Cited by 6 | Viewed by 2366

Abstract

The main branch of vitamin D₃ metabolism involves several hydroxylation reactions to obtain mono-, di- and trihydroxylated metabolites, including the circulating and active forms—25(OH)D₃ and 1,25(OH)₂D₃, respectively. However, most clinical trials strictly target the determination of 25(OH)D₃ to offer a view of the metabolic status of vitamin D₃. Due to the growing interest in expanding this restricted view, we have developed a method for measuring vitamin D₃ metabolism by determination of vitamin D₃, 25(OH)D₃, 24,25(OH)₂D₃, 1,25(OH)₂D₃ and 1,24,25(OH)₃D₃ in human plasma. The method was based on SPE–LC–MS/MS with a large volume injection of human plasma (240 µL). Detection of di- and trihydroxymetabolites, found at the picogram per milliliter level, was attained by the combined action of high preconcentration and clean-up effects. The method allows obtaining information about ratios such as the known vitamin D metabolite ratio (24,25(OH)₂D₃/25(OH)D₃), which can provide complementary views of vitamin D₃ metabolic status. The method was applied to a cohort of obese patients and a reference cohort of healthy volunteers to find metabolic correlations between target analytes as well as differences as a function of vitamin D levels within and between cohorts. Full article

(This article belongs to the Special Issue Chromatographic Analysis of Biological Samples)

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9 pages, 1130 KiB

Open AccessCommunication

Phytochemical Constituents Identified from the Aerial Parts of Lespedeza cuneata and Their Effects on Lipid Metabolism during Adipocyte Maturation

by Heesun Kang, Min Jeong Yoo, Sang Ah Yi, Tae Wan Kim, Ji Won Ha, Myung Woo Na, Kun Hee Park, Seon-Hee Kim, Jeung-Whan Han, Tae Su Jang and Ki Hyun Kim

Separations 2021, 8(11), 203; https://doi.org/10.3390/separations8110203 - 3 Nov 2021

Cited by 2 | Viewed by 2130

Abstract

Lespedeza cuneata, belonging to Fabaceae, is well-known as Chinese bushclover, and it has been used in traditional folk medicines for the treatment of disorders, such as diabetes, hematuria, and insomnia. As part of continuing research projects to discover interesting natural compounds with biological activities from Korean medicinal plants, the phytochemical investigation of L. cuneata resulted in the isolation of five chemical constituents: α-tocopherol (1), 7a-methoxy-α-tocopherol (2), 13(R)-hydroxy-octadeca-(9Z,11E,15Z)-trien-oic acid (3), α-dimorphecolic acid (4), and lupeol (5). The structural determination of the isolated compounds was elucidated from data gathered through nuclear magnetic resonance (NMR) spectroscopy and liquid chromatography–mass spectrometry (LC/MS). Until now, this study is the first to report these five compounds from the plant L. cuneata. Moreover, these isolated compounds (1–5) were evaluated for their anti-adipogenesis effects and their role in lipid metabolism during adipocyte maturation. As a result, the upregulation of mRNA expression levels of Fabp4 from 3T3-L1 pre-adipocytes treated with compounds 3 and 4 demonstrated that these compounds efficiently induced adipocyte differentiation. Furthermore, compounds 3 and 4 were found to regulate lipid metabolism by the induction of lipolytic and of lipogenic gene expressions. Therefore, experimental data from these findings supported that the compounds 3 and 4 induce the adipogenesis of 3T3-L1 pre-adipocytes and regulate lipid metabolism. Full article

(This article belongs to the Special Issue Chromatographic Analysis of Biological Samples)

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18 pages, 5863 KiB

Open AccessArticle

Determination and Chemometrics-Assisted Comparative Analysis of Active Components in Different Tissue of Rana chensinensis

by Jianqiu Zhang, Zhongyao Wang, Shihan Wang, Changli Zhang, Nan Li, Dongliang Xu, Yong Yang and Yongsheng Wang

Separations 2021, 8(10), 164; https://doi.org/10.3390/separations8100164 - 28 Sep 2021

Cited by 1 | Viewed by 1903

Abstract

In this study, the chemical composition of different tissues of Rana temporaria chensinensis David derived from the same individual was analyzed by comparative approach. First, pre-column derivatization combined with high performance liquid chromatography (HPLC) was established to determine the content of 1-methyl hydantoin in samples, which used S1–S5 samples. The results indicated that 1-methyl hydantoin was determined in Oviductus Ranae (OR), Rana chensinensis ovum (RCO), Rana chensinensis meat (RCM), and Rana chensinensis skin (RCS), except for Rana chensinensis bone (RCB). Moreover, the content of it in RCS was the highest. In addition, the contents of six polyunsaturated fatty acids (PUFAs) in different tissues of Rana chensinensis were measured by HPLC, including eicosapentaenoic acid (EPA), α-linolenic acid (ALA), docosahexaenoic acid (DHA), arachidonic acid (ARA), linoleic acid (LA) and oleic acid (OA). The results indicated that OR, RCO, RCM, RCS, and RCB all contained the above six PUFAs. With the aid of chemometrics methods, the results of principal component analysis (PCA), hierarchical cluster analysis (HCA), and orthogonal partial least squares discriminant analysis (OPLS-DA) combined with the sequencing results of the total PUFAs content of each sample, showed that different tissues of Rana chensinensis could be divided into four categories, and the RCO sample was divided into one category because of the highest PUFAs content, which was a good source of PUFA. For comparison, OR and other tissue from the perspective of PUFAs, we also established OPLS-DA models of them. It could be found that the RCM was the most similar to the OR in the diversity and content of PUFAs. This study provided a theoretical basis for the further development and utilization of RCO, RCM, RCS, and RCB as by-products of OR. Full article

(This article belongs to the Special Issue Chromatographic Analysis of Biological Samples)

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15 pages, 1833 KiB

Open AccessFeature PaperArticle

Efficient Sub-1 Minute Analysis of Selected Biomarker Catecholamines by Core-Shell Hydrophilic Interaction Liquid Chromatography (HILIC) with Nanomolar Detection at a Boron-Doped Diamond (BDD) Electrode

by Majidah Alsaeedi, Huda Alghamdi, Phyllis E. Hayes, Anna M. Hogan and Jeremy D. Glennon

Separations 2021, 8(8), 124; https://doi.org/10.3390/separations8080124 - 18 Aug 2021

Cited by 2 | Viewed by 2546

Abstract

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

(This article belongs to the Special Issue Chromatographic Analysis of Biological Samples)

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19 pages, 759 KiB

Open AccessFeature PaperEditor’s ChoiceArticle

Andean Blueberry of the Genus Disterigma: A High-Resolution Mass Spectrometric Approach for the Comprehensive Characterization of Phenolic Compounds

by Sara Elsa Aita, Anna Laura Capriotti, Chiara Cavaliere, Andrea Cerrato, Benedetta Giannelli Moneta, Carmela Maria Montone, Susy Piovesana and Aldo Laganà

Separations 2021, 8(5), 58; https://doi.org/10.3390/separations8050058 - 2 May 2021

Cited by 24 | Viewed by 3457

Abstract

Wild neotropical blueberries, endemic of Central and South American areas, are promising yet still undisclosed sources of bioactive compounds. Most research studies have addressed wild and cultivated blueberries from Europe and North America, despite the extremely wide variety of wild neotropical species. In the present paper, for the first time, the phenolic composition of Disterigma alaternoides was investigated through ultra-high-performance liquid chromatography coupled to high-resolution mass-spectrometric analysis followed by accurate data analysis and compound validation with a dedicated structure-based workflow. D. alaternoides, which belongs to a closely related genus to that of the common blueberry, grows exclusively in the Andean regions over 2000 above sea level. Thanks to the dedicated analytical platform, 249 phenolic compounds were tentatively identified, including several anthocyanins, flavonoids, phenolic acids, and proanthocyanidins. Thenature and heterogeneity of identified phenolic compounds demonstrate once more the need for a more profound knowledge of such still uncharted matrices. Full article

(This article belongs to the Special Issue Chromatographic Analysis of Biological Samples)

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15 pages, 2281 KiB

Open AccessArticle

Protocol Optimization of Proteomic Analysis of Korean Ginseng (Panax ginseng Meyer)

by Clarissa Braccia, Bhakti Prinsi, Mara Colzani, Alessandra A. Altomare, Luca Espen, Yoon-Mi Lee, Giancarlo Aldini and Kyung-Jin Yeum

Separations 2021, 8(4), 53; https://doi.org/10.3390/separations8040053 - 19 Apr 2021

Cited by 1 | Viewed by 3005

Abstract

The benefits of ginseng have been mainly attributed to its triterpenoids, called ginsenosides. Recent genome sequencing of the Panax ginseng has paved the way for in-depth proteomic studies of this medicinal plant. The current study was conducted to deepen the proteomic information on the root proteome of Korean ginseng. Proteomic workflow was optimized by testing two different strategies, characterized by the phenol extraction procedure, the presence or the absence of SDS-PAGE fractionation step, and nano-scale liquid chromatographic tandem mass spectrometry (nLC-MS/MS) analysis. The results highlighted an evident improvement of proteome extraction by the combination of phenol extraction with SDS-PAGE before the nLC-MS/MS analysis. In addition, a dramatic impact of the steaming process (the treatment to produce red ginseng from ginseng) on protein properties was observed. Overall, the analyses of Korean ginseng permitted the characterization of a total of 2412 proteins. A large number of identified proteins belonged to the functional categories of protein and carbon/energy metabolism (22.4% and 14.6%, respectively). The primary and secondary metabolisms are major metabolic pathways, which emerged from the proteomic analysis. In addition, a large number of proteins known to play an important role in response to (a)biotic stresses were also identified. The current proteomic study not only confirmed the previous transcriptomic and proteomic reports but also extended proteomic information, including the main metabolic pathways involved in Korean ginseng. Full article

(This article belongs to the Special Issue Chromatographic Analysis of Biological Samples)

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19 pages, 5660 KiB

Open AccessArticle

Chromatographic Profiling with Machine Learning Discriminates the Maturity Grades of Nicotiana tabacum L. Leaves

by Yi Chen, Miao Tian, Gaokun Zhao, Hongmei Lu, Zhimin Zhang and Congming Zou

Separations 2021, 8(1), 9; https://doi.org/10.3390/separations8010009 - 19 Jan 2021

Cited by 3 | Viewed by 3049

Abstract

Nicotiana tabacum L. (NTL) is an important agricultural and economical crop. Its maturity is one of the key factors affecting its quality. Traditionally, maturity is discriminated visually by humans, which is subjective and empirical. In this study, we concentrated on detecting as many compounds as possible in NTL leaves from different maturity grades using ultra-performance liquid chromatography ion trap time-of-flight mass spectrometry (UPLC-IT-TOF/MS). Then, the low-dimensional embedding of LC-MS dataset by t-distributed stochastic neighbor embedding (t-SNE) clearly showed the separation of the leaves from different maturity grades. The discriminant models between different maturity grades were established using orthogonal partial least squares discriminant analysis (OPLS-DA). The quality metrics of the models are R²Y = 0.939 and Q² = 0.742 (unripe and ripe), R²Y = 0.900 and Q² = 0.847 (overripe and ripe), and R²Y = 0.972 and Q² = 0.930 (overripe and unripe). The differential metabolites were screened by their variable importance in projection (VIP) and p-Values. The existing tandem mass spectrometry library of plant metabolites, the user-defined library of structures, and MS-FINDER were combined to identify these metabolites. A total of 49 compounds were identified, including 12 amines, 14 lipids, 10 phenols, and 13 others. The results can be used to discriminate the maturity grades of the leaves and ensure their quality. Full article

(This article belongs to the Special Issue Chromatographic Analysis of Biological Samples)

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