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Analysis of Drugs in Biological Samples through Liquid Chromatography

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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 (1 August 2021) | Viewed by 32097

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Special Issue Editors

Dr. Anna Petruczynik

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Department of Inorganic Chemistry, Medical University of Lublin, Chodźki 4a, 20-093 Lublin, Poland
Interests: liquid chromatography; optimization of chromatographic systems for separation of ionic compounds; quantitative analysis of ionic compounds in biological samples
Special Issues, Collections and Topics in MDPI journals

Prof. Dr. Tomasz Tuzimski

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Department of Physical Chemistry, Medical University of Lublin, Chodźki 4a, 20-093 Lublin, Poland
Interests: liquid chromatography with modern detection techniques; sample preparation; analysis of xenobiotics in various biological samples; analysis of ionic compounds in plant extracts; biological activity of plant extracts
Special Issues, Collections and Topics in MDPI journals

Special Issue Information

Dear Colleagues,

Drug analysis is a very important aspect of scientific research. Drugs are very diverse compounds in terms of structure and chemical properties. The action of these compounds is highly dose-dependent. Each drug has a therapeutic range, and the existence of drug at concentrations lower than the minimum level causes weak beneficial effects for patients, while concentrations above than maximum limit cause side effects, which may be dangerous for patients. Today, more and more frequently therapeutic drug monitoring is applied in clinical practice. The establishment of methodologies for drug monitoring in biological matrices is essential for patients’ safety. Thus, highly efficient techniques involved in the extraction and enrichment, separation, and sensitive and selective detection are required for the reliable determination of drugs in complex biological samples.

To determine the concentrations of drugs and their metabolites, several methods have been developed in which selection depends on the complexity of the sample and the natures of the analytes and the matrices. Biological matrices are complex and often contain proteins, lipids, salts, acids, bases, and various other organic and inorganic compounds with similar properties to the analytes, which often exist at low concentrations in samples. Most methods for analyzing drugs belonging to various therapeutic classes in different biological samples are based on combining a very efficient separation technique, including high-performance liquid chromatography (HPLC), ultrahigh-performance liquid chromatography (UPLC), and high-performance thin-layer chromatography (HPTLC) with a sensitive detection method. Liquid chromatography is one of the most efficient and robust specific technique due to the merits of convenience, simple operation, strong separation ability, and wide sample application. High-performance liquid chromatography (HPLC) with UV-VIS, fluorescence, diode array (DAD), and mass spectrometry (LC-MS) or tandem mass spectrometry (LC–MS/MS) detection methods has often been applied for the analysis of different drugs in various biological samples such as serum, plasma, urine, saliva, breast milk, and other biological samples. Liquid chromatography is currently widely used for the analysis of drugs and dosage forms with respect to quality control, quantitative determination of active ingredients and impurities, monitoring drug blood and other tissue concentration in patients, and bioequivalence assessment. The chromatography column plays an important role in the analytes’ separation process. Therefore, a kind of stationary phase and its parameters, such as inner diameter and sorbent particle size, are very important. The composition of a mobile phase plays an equally important role. The mobile phase’s composition in liquid chromatography is selected based on the chromatographic behavior of the investigated analytes and impurities and the detection method.

Sample preparation before chromatographic analysis is an important stage in the analysis of drugs in biological samples. It enables accompanying components to be removed and the drug to be concentrated. The choice of a sample preparation method before chromatographic analysis is closely related to the properties of the investigated drugs, metabolites, and matrices.

We cordially invite researchers working in this field to contribute original research articles, short communications, and critical review articles. Short papers on one compound will also be welcome.

Dr. Anna Petruczynik (Ph.D., Adjunct Professor)
Prof. Dr. Tomasz Tuzimski (Ph.D., Adjunct Professor)
Guest Editors

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Keywords

Drug analysis
Liquid chromatography
Detection techniques
Therapeutic approaches
Sample preparation

Published Papers (8 papers)

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Research

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23 pages, 2345 KiB

Open AccessArticle

Repurposing of Omarigliptin as a Neuroprotective Agent Based on Docking with A_2A Adenosine and AChE Receptors, Brain GLP-1 Response and Its Brain/Plasma Concentration Ratio after 28 Days Multiple Doses in Rats Using LC-MS/MS

by Bassam M. Ayoub, Haidy E. Michel, Shereen Mowaka, Moataz S. Hendy and Mariam M. Tadros

Molecules 2021, 26(4), 889; https://0-doi-org.brum.beds.ac.uk/10.3390/molecules26040889 - 08 Feb 2021

Cited by 11 | Viewed by 4001

Abstract

The authors in the current work suggested the potential repurposing of omarigliptin (OMR) for neurodegenerative diseases based on three new findings that support the preliminary finding of crossing BBB after a single dose study in the literature. The first finding is the positive results of the docking study with the crystal structures of A_2A adenosine (A2AAR) and acetylcholine esterase (AChE) receptors. A2AAR is a member of non-dopaminergic GPCR superfamily receptor proteins and has essential role in regulation of glutamate and dopamine release in Parkinson’s disease while AChE plays a major role in Alzheimer’s disease as the primary enzyme responsible for the hydrolytic metabolism of the neurotransmitter acetylcholine into choline and acetate. Docking showed that OMR perfectly fits into A2AAR binding pocket forming a distinctive hydrogen bond with Threonine 256. Besides other non-polar interactions inside the pocket suggesting the future of the marketed anti-diabetic drug (that cross BBB) as a potential antiparkinsonian agent while OMR showed perfect fit inside AChE receptor binding site smoothly because of its optimum length and the two fluorine atoms that enables quite lean fitting. Moreover, a computational comparative study of OMR docking, other 12 DPP-4 inhibitors and 11 SGLT-2 inhibitors was carried out. Secondly, glucagon-like peptide-1 (GLP-1) concentration in rats’ brain tissue was determined by the authors using sandwich GLP-1 ELISA kit bio-analysis to ensure the effect of OMR after the multiple doses’ study. Brain GLP-1 concentration was elevated by 1.9-fold following oral multiple doses of OMR (5 mg/kg/day, p.o. for 28 days) as compared to the control group. The third finding is the enhanced BBB crossing of OMR after 28 days of multiple doses that had been studied using LC-MS/MS method with enhanced liquid–liquid extraction. A modified LC-MS/MS method was established for bioassay of OMR in rats’ plasma (10–3100 ng/mL) and rats’ brain tissue (15–2900 ng/mL) using liquid–liquid extraction. Alogliptin (ALP) was chosen as an internal standard (IS) due to its LogP value of 1.1, which is very close to the LogP of OMR. Extraction of OMR from samples of both rats’ plasma and rats’ brain tissue was effectively achieved with ethyl acetate as the extracting solvent after adding 1N sodium carbonate to enhance the drug migration, while choosing acetonitrile to be the diluent solvent for the IS to effectively decrease any emulsion between the layers in the stated method of extraction. Validation results were all pleasing including good stability studies with bias of value below 20%. Concentration of OMR in rats’ plasma were determined after 2 h of the latest dose from 28 days multiple doses, p.o, 5 mg/kg/day. It was found to be 1295.66 ± 684.63 ng/mL estimated from the bio-analysis regression equation. OMR passed through the BBB following oral administration and exhibited concentration of 543.56 ± 344.15 ng/g in brain tissue, taking in consideration the dilution factor of 10. The brain/plasma concentration ratio of 0.42 (543.56/1295.66) was used to illustrate the penetration power through the BBB after the multiple doses for 28 days. Results showed that OMR passed through the BBB more effectively in the multiple dose study as compared to the previously published single dose study by the authors. Thus, the present study suggests potential repositioning of OMR as antiparkinsonian agent that will be of interest for researchers interested in neurodegenerative diseases. Full article

(This article belongs to the Special Issue Analysis of Drugs in Biological Samples through Liquid Chromatography)

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11 pages, 1312 KiB

Open AccessFeature PaperArticle

High-Performance Liquid Chromatography-Tandem Mass Spectrometry for Buprenorphine Evaluation in Plasma—Application to Pharmacokinetic Studies in Rabbits

by Marta Tikhomirov, Błażej Poźniak and Tomasz Śniegocki

Molecules 2021, 26(2), 437; https://0-doi-org.brum.beds.ac.uk/10.3390/molecules26020437 - 15 Jan 2021

Cited by 6 | Viewed by 1856

Abstract

The precise and reliable determination of buprenorphine concentration is fundamental in certain medical or research applications, particularly in pharmacokinetic studies of this opioid. The main challenge is, however, the development of an analytical method that is sensitive enough, as the detected in vivo concentrations often fall in very low ranges. Thus, in this study we aimed at developing a sensitive, repeatable, cost-efficient, and easy HPLC analytical protocol for buprenorphine in rabbit plasma. In order to obtain this, the HPLC-MS² system was used to elaborate and validate the method for samples purified with liquid-liquid extraction. Fragment ions 468.6→396.2 and 468.6→414.2 were monitored, and the method resulted in a high repeatability and reproducibility and a limit of quantification of 0.25 µg/L with a recovery of 98.7–109.0%. The method was linear in a range of 0.25–2000 µg/L. The suitability of the analytical procedure was tested in rabbits in a pilot pharmacokinetic study, and it was revealed that the method was suitable for comprehensively describing the pharmacokinetic profile after buprenorphine intravenous administration at a dose of 300 µg/kg. Thus, the method suitability for pharmacokinetic application was confirmed by both the good validation results of the method and successful in vivo tests in rabbits. Full article

(This article belongs to the Special Issue Analysis of Drugs in Biological Samples through Liquid Chromatography)

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16 pages, 1395 KiB

Open AccessArticle

HPLC Determination of Imidazoles with Variant Anti-Infective Activity in Their Dosage Forms and Human Plasma

by Oday T. Ali, Wafaa S. Hassan, Ahdab N. Khayyat, Ahmad J. Almalki and Mahmoud M. Sebaiy

Molecules 2021, 26(1), 129; https://0-doi-org.brum.beds.ac.uk/10.3390/molecules26010129 - 30 Dec 2020

Cited by 3 | Viewed by 2983

Abstract

A suitable HPLC method has been selected and validated for rapid simultaneous separation and determination of four imidazole anti-infective drugs, secnidazole, omeprazole, albendazole, and fenbendazole, in their final dosage forms, in addition to human plasma within 5 min. The method suitability was derived from the superiority of using the environmentally benign solvent, methanol over acetonitrile as a mobile phase component in respect of safety issues and migration times. Separation of the four anti-infective drugs was performed on a Thermo Scientific^® BDS Hypersil C₈ column (5 µm, 2.50 × 4.60 mm) using a mobile phase consist of MeOH: 0.025 M KH₂PO₄ (70:30, v/v) adjusted to pH 3.20 with ortho-phosphoric acid at room temperature. The flow rate was 1.00 mL/min and maximum absorption was measured with UV detector set at 300 nm. Limits of detection were reported to be 0.41, 0.13, 0.18, and 0.15 µg/mL for secnidazole, omeprazole, albendazole, and fenbendazole, respectively, showing a high degree of the method sensitivity. The method of analysis was validated according to Food and Drug Administration (FDA)guidelines for the determination of the drugs, either in their dosage forms with highly precise recoveries, or clinically in human plasma, especially regarding pharmacokinetic and bioequivalence studies. Full article

(This article belongs to the Special Issue Analysis of Drugs in Biological Samples through Liquid Chromatography)

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16 pages, 1439 KiB

Open AccessFeature PaperArticle

Sensitive Analysis of Idarubicin in Human Urine and Plasma by Liquid Chromatography with Fluorescence Detection: An Application in Drug Monitoring

by Olga Maliszewska, Natalia Treder, IIona Olędzka, Piotr Kowalski, Natalia Miękus, Tomasz Bączek, Wojciech Rodzaj, Ewa Bień, Małgorzata Anna Krawczyk and Alina Plenis

Molecules 2020, 25(24), 5799; https://0-doi-org.brum.beds.ac.uk/10.3390/molecules25245799 - 09 Dec 2020

Cited by 2 | Viewed by 2129

Abstract

A new approach for the sensitive, robust and rapid determination of idarubicin (IDA) in human plasma and urine samples based on liquid chromatography with fluorescence detection (LC-FL) was developed. Satisfactory chromatographic separation of the analyte after solid-phase extraction (SPE) was performed on a Discovery HS C18 analytical column using a mixture of acetonitrile and 0.1% formic acid in water as the mobile phase in isocratic mode. IDA and daunorubicin hydrochloride used as an internal standard (I.S.) were monitored at the excitation and emission wavelengths of 487 and 547 nm, respectively. The method was validated according to the FDA and ICH guidelines. The linearity was confirmed in the range of 0.1–50 ng/mL and 0.25–200 ng/mL, while the limit of detection (LOD) was 0.05 and 0.125 ng/mL in plasma and urine samples, respectively. The developed LC-FL method was successfully applied for drug determinations in human plasma and urine after oral administration of IDA at a dose of 10 mg to a patient with highly advanced alveolar rhabdomyosarcoma (RMA). Moreover, the potential exposure to IDA present in both fluids for healthcare workers and the caregivers of patients has been evaluated. The present LC-FL method can be a useful tool in pharmacokinetic and clinical investigations, in the monitoring of chemotherapy containing IDA, as well as for sensitive and reliable IDA quantitation in biological fluids. Full article

(This article belongs to the Special Issue Analysis of Drugs in Biological Samples through Liquid Chromatography)

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12 pages, 1166 KiB

Open AccessArticle

Development and Validation of High-Throughput Bioanalytical Liquid Chromatography-Tandem Mass Spectrometry (LC-MS/MS) Method for the Quantification of Newly Synthesized Antitumor Carbonic Anhydrase Inhibitors in Human Plasma

by Ahmed M. Abdel-Megied, Wagdy M. Eldehna, Mohamed A. Abdelrahman and Fawzy A. Elbarbry

Molecules 2020, 25(23), 5753; https://0-doi-org.brum.beds.ac.uk/10.3390/molecules25235753 - 06 Dec 2020

Cited by 1 | Viewed by 2325

Abstract

In the present study, a sensitive and fully validated bioanalytical high-performance liquid chromatography-tandem mass spectrometry (LC-MS/MS) method has been developed for the quantitative determination of three newly synthesized carbonic anhydrases inhibitors (CAIs) with potential antitumor activity in human plasma. The analytes and the internal standard (IS) were extracted using 1.5 mL acetonitrile from only 450 µL aliquots of human plasma to achieve the desired protein precipitation. Chromatographic separations were achieved on Phenomenex Kinetex^® C₁₈ column (100 × 4.6 mm, 2.6 µm) using a binary gradient elution mode with a run time of less than 6 min. The mobile phase consisted of solvent (A): 0.1% formic acid in 50% methanol and solvent B: 0.1% formic acid in acetonitrile (30:70, v/v), pumped at a flow rate of 0.8 mL/min. Detection was employed using triple quadrupole tandem mass spectrometer (API 3500) equipped with an electrospray ionization (ESI) source in the positive ion mode. Multiple reaction monitoring (MRM) mode was selected for quantitation through monitoring the precursor-to-parent ion transition at m/z 291.9 → 173.0, m/z 396.9 → 225.1, m/z 388.9 → 217.0, and m/z 146.9 → 91.0 for AW-9a, WES-1, WES-2, and Coumarin (IS), respectively. Linearity was computed using the weighted least-squares linear regression method (1/x²) over a concentration range of 1–1000, 2.5–800, and 5–500 ng/mL for AW-9a, WES-1, and WES-2; respectively. The bioanalytical LC-MS/MS method was fully validated as per U.S. Food and Drug Administration (FDA) guidelines with all respect to linearity, accuracy, precision, carry-over, selectivity, dilution integrity, and stability. The proposed LC-MS/MS method was applied successfully for the determination of all investigated drugs in spiked human plasma with no significant matrix effect, which is a crucial cornerstone in further therapeutic drug monitoring of newly developed therapeutic agents. Full article

(This article belongs to the Special Issue Analysis of Drugs in Biological Samples through Liquid Chromatography)

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11 pages, 2957 KiB

Open AccessArticle

Bioanalytical Method Using Ultra-High-Performance Liquid Chromatography Coupled with High-Resolution Mass Spectrometry (UHPL-CHRMS) for the Detection of Metformin in Human Plasma

by Ye-Ji Kang, Hyeon-Cheol Jeong, Tae-Eun Kim and Kwang-Hee Shin

Molecules 2020, 25(20), 4625; https://0-doi-org.brum.beds.ac.uk/10.3390/molecules25204625 - 11 Oct 2020

Cited by 8 | Viewed by 2687

Abstract

Metformin is the first-line medicine for the treatment of type 2 diabetes. Drug interactions between metformin and other drugs, food, or beverages cannot only cause changes in the pharmacokinetic profiles but also affect the efficacy of metformin. The purpose of this study was to develop a rapid and reliable bioanalytical method for the detection of plasma metformin concentration in humans. To remove interfering substances in plasma, acidified acetonitrile (acetonitrile containing 0.1% formic acid) was added to samples. Ultra-high-performance liquid chromatography (UHPLC) coupled with high resolution mass spectrometry (HRMS) was used to analyze metformin and its internal standard (metformin-d6). Analyte separation was performed on a BEH HILIC analytical column (100 × 2.1 mm, 1.7 μm) using a gradient elution of 0.1% formic acid (A) and acetonitrile with 0.1% formic acid (B). The total chromatographic run time was 2 min. The developed method was validated for its linearity, accuracy and precision, selectivity (signal of interfering substance; analyte, lower limit of quantification (LLOQ) ≤ 20%; IS, IS ≤ 5%), sensitivity (LLOQ, 5 ng/mL; S/N ratio ≥ 10), stability (low quality control (LQC, 15 ng/mL), 2.95–14.19%; high quality control (HQC, 1600 ng/mL), −9.49–15.10%), dilution integrity (diluted QC (4000 ng/mL); 10-folds diluted QC (400 ng/mL); 5-folds diluted QC (800 ng/mL); accuracy, 81.30–91.98%; precision, ≤4.47%), carry-over (signal of double blank; analyte, LLOQ ≤20%; IS, IS ≤5%), and matrix effect (LQC, 10.109%; HQC, 12.271%) under various conditions. The constructed calibration curves were shown linear in the concentration range of 5–2000 ng/mL, with within- and between-run precision values of <8.19% and accuracy in the range of 91.13–105.25%. The plasma metformin concentration of 16 healthy subjects was successfully measured by applying the validated bioanalytical method. Full article

(This article belongs to the Special Issue Analysis of Drugs in Biological Samples through Liquid Chromatography)

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Review

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29 pages, 1333 KiB

Open AccessReview

New Methods Used in Pharmacokinetics and Therapeutic Monitoring of the First and Newer Generations of Antiepileptic Drugs (AEDs)

by Karina Sommerfeld-Klatta, Barbara Zielińska-Psuja, Marta Karaźniewcz-Łada and Franciszek K. Główka

Molecules 2020, 25(21), 5083; https://0-doi-org.brum.beds.ac.uk/10.3390/molecules25215083 - 02 Nov 2020

Cited by 21 | Viewed by 4764

Abstract

The review presents data from the last few years on bioanalytical methods used in therapeutic drug monitoring (TDM) of the 1st–3rd generation and the newest antiepileptic drug (AEDs) cenobamate in patients with various forms of seizures. Chemical classification, structure, mechanism of action, pharmacokinetic data and therapeutic ranges for total and free fractions and interactions were collected. The primary data on bioanalytical methods for AEDs determination included biological matrices, sample preparation, dried blood spot (DBS) analysis, column resolution, detection method, validation parameters, and clinical utility. In conclusion, the most frequently described method used in AED analysis is the LC-based technique (HPLC, UHPLC, USLC) combined with highly sensitive mass detection or fluorescence detection. However, less sensitive UV is also used. Capillary electrophoresis and gas chromatography have been rarely applied. Besides the precipitation of proteins or LLE, an automatic SPE is often a sample preparation method. Derivatization was also indicated to improve sensitivity and automate the analysis. The usefulness of the methods for TDM was also highlighted. Full article

(This article belongs to the Special Issue Analysis of Drugs in Biological Samples through Liquid Chromatography)

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42 pages, 13818 KiB

Open AccessReview

Determination and Identification of Antibiotic Drugs and Bacterial Strains in Biological Samples

by Katarzyna Pauter, Małgorzata Szultka-Młyńska and Bogusław Buszewski

Molecules 2020, 25(11), 2556; https://0-doi-org.brum.beds.ac.uk/10.3390/molecules25112556 - 31 May 2020

Cited by 25 | Viewed by 8892

Abstract

Antibiotics were initially natural substances. However, nowadays, they also include synthetic drugs, which show their activity against bacteria, killing or inhibiting their growth and division. Thanks to these properties, many antibiotics have quickly found practical application in the fight against infectious diseases such as tuberculosis, syphilis, gastrointestinal infections, pneumonia, bronchitis, meningitis and septicemia. Antibiotic resistance is currently a detrimental problem; therefore, in addition to the improvement of antibiotic therapy, attention should also be paid to active metabolites in the body, which may play an important role in exacerbating the existing problem. Taking into account the clinical, cognitive and diagnostic purposes of drug monitoring, it is important to select an appropriate analytical method that meets all the requirements. The detection and identification of the microorganism responsible for the infection is also an essential factor in the implementation of appropriate antibiotic therapy. In recent years, clinical microbiology laboratories have experienced revolutionary changes in the way microorganisms are identified. The MALDI-TOF MS technique may be interesting, especially in some areas where a quick analysis is required, as is the case with clinical microbiology. This method is not targeted, which means that no prior knowledge of the infectious agent is required, since identification is based on a database match. Full article

(This article belongs to the Special Issue Analysis of Drugs in Biological Samples through Liquid Chromatography)

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