bims-metlip 2022-11-27 papers

bims-metlip

Biomed News

on Methods and protocols in metabolomics and lipidomics

Issue of 2022‒11‒27
27 papers selected by
Sofia Costa
Matterworks

The critical role that spectral libraries play in capturing the metabolomics community knowledge.
Compound Identification Strategies in Mass Spectrometry-Based Metabolomics and Pharmacometabolomics.
Unified-Hydrophilic-Interaction/Anion-Exchange Liquid Chromatography Mass Spectrometry (Unified-HILIC/AEX/MS): A Single-Run Method for Comprehensive and Simultaneous Analysis of Polar Metabolome.
Determination of 19 Steroid Hormones in Human Serum and Urine Using Liquid Chromatography-Tandem Mass Spectrometry.
Branched medium-chain fatty acid profiling and enantiomer separation of anteiso-forms of teicoplanin fatty acyl side chain RS3 using UHPLC-MS/MS with polysaccharide columns.
A sensitive and accurate GC-MS method for analyzing microbial metabolites short chain fatty acids and their hydroxylated derivatives in newborn fecal samples.
Lipid Serum Profiling of Boar-Tainted and Untainted Pigs Using GC×GC-TOFMS: An Exploratory Study.
An Optimised Monophasic Faecal Extraction Method for LC-MS Analysis and Its Application in Gastrointestinal Disease.
The Development of New Methodology for Determination of Vincristine (VCR) in Human Serum Using LC-MS/MS-Based Method for Medical Diagnostics.
LC-HRMS/MS-Based Metabolomics Approaches Applied to the Detection of Antifungal Compounds and a Metabolic Dynamic Assessment of Orchidaceae.
Applications of liquid chromatography-mass spectrometry based metabolomics in predictive and personalized medicine.
MZA: A Data Conversion Tool to Facilitate Software Development and Artificial Intelligence Research in Multidimensional Mass Spectrometry.
Use of tandem carbon microfiber columns for on-line fractionation strategy of reducing ion suppression effects in electrospray ionization-mass spectrometry.
The Exploration of Microbial Natural Products and Metabolic Interaction Guided by Mass Spectrometry Imaging.
How searching against multiple libraries can lead to biased results in GC/MS-based metabolomics.
High-Resolution Ion Mobility Spectrometry Mass Spectrometry for Isomeric Separation of Prostanoids after Girard's reagent T Derivatization.
Evaluating Different Extraction Approaches for GC-MS Based Metabolomics Analysis of the Giant Pandas' Fur.
Comprehensive Steroid Assay with Non-Targeted Analysis Using Liquid Chromatography Ion Mobility Mass Spectrometry.
Simultaneous Determination of 11 Mycotoxins in Maize via Multiple-Impurity Adsorption Combined with Liquid Chromatography-Tandem Mass Spectrometry.
Metabolomics- and systems-biology-guided discovery of metabolite lead compounds and druggable targets.
High-Throughput Metabolic Soft-Spot Identification in Liver Microsomes by LC/UV/MS: Application of a Single Variable Incubation Time Approach.
Chemical shift variations in common metabolites.
Development and multicenter validation of an LC-MS-based bioanalytical method for antisense therapeutics.
An LC-MS/MS method for the simultaneous determination of 18 antibacterial drugs in human plasma and its application in therapeutic drug monitoring.
Development of simple and rapid method for Emicizumab quantification by LC-MS/MS in human plasma.
Ganglioside analysis in body fluids by liquid-phase separation techniques hyphenated to mass spectrometry.
Degradation Pathways and Impurity Profiling of Anti-Cancer Drug Apalutamide by HPLC and LC-MS/MS and Separation of Impurities using Design of Experiments.

Metabolomics. 2022 Nov 19. 18(12): 94

The critical role that spectral libraries play in capturing the metabolomics community knowledge.

Wout Bittremieux, Mingxun Wang, Pieter C Dorrestein.

  BACKGROUND: Spectral library searching is currently the most common approach for compound annotation in untargeted metabolomics. Spectral libraries applicable to liquid chromatography mass spectrometry have grown in size over the past decade to include hundreds of thousands to millions of mass spectra and tens of thousands of compounds, forming an essential knowledge base for the interpretation of metabolomics experiments.AIM OF REVIEW: We describe existing spectral library resources, highlight different strategies for compiling spectral libraries, and discuss quality considerations that should be taken into account when interpreting spectral library searching results. Finally, we describe how spectral libraries are empowering the next generation of machine learning tools in computational metabolomics, and discuss several opportunities for using increasingly accessible large spectral libraries.
KEY SCIENTIFIC CONCEPTS OF REVIEW: This review focuses on the current state of spectral libraries for untargeted LC-MS/MS based metabolomics. We show how the number of entries in publicly accessible spectral libraries has increased more than 60-fold in the past eight years to aid molecular interpretation and we discuss how the role of spectral libraries in untargeted metabolomics will evolve in the near future.

Keywords:  Compound identification; Mass spectrometry; Spectral library; Untargeted metabolomics

DOI:  https://doi.org/10.1007/s11306-022-01947-y
Handb Exp Pharmacol. 2022 Nov 22.

Compound Identification Strategies in Mass Spectrometry-Based Metabolomics and Pharmacometabolomics.

Rylan Hissong, Kendra R Evans, Charles R Evans.

  The metabolome is composed of a vast array of molecules, including endogenous metabolites and lipids, diet- and microbiome-derived substances, pharmaceuticals and supplements, and exposome chemicals. Correct identification of compounds from this diversity of classes is essential to derive biologically relevant insights from metabolomics data. In this chapter, we aim to provide a practical overview of compound identification strategies for mass spectrometry-based metabolomics, with a particular eye toward pharmacologically-relevant studies. First, we describe routine compound identification strategies applicable to targeted metabolomics. Next, we discuss both experimental (data acquisition-focused) and computational (software-focused) strategies used to identify unknown compounds in untargeted metabolomics data. We then discuss the importance of, and methods for, assessing and reporting the level of confidence of compound identifications. Throughout the chapter, we discuss how these steps can be implemented using today's technology, but also highlight research underway to further improve accuracy and certainty of compound identification. For readers interested in interpreting metabolomics data already collected, this chapter will supply important context regarding the origin of the metabolite names assigned to features in the data and help them assess the certainty of the identifications. For those planning new data acquisition, the chapter supplies guidance for designing experiments and selecting analysis methods to enable accurate compound identification, and it will point the reader toward best-practice data analysis and reporting strategies to allow sound biological and pharmacological interpretation.

Keywords:  Compound identification; Identification confidence; LC-MS; MS/MS search; Metabolomics; Molecular formula assignment

DOI:  https://doi.org/10.1007/164_2022_617
Anal Chem. 2022 Nov 25.

Unified-Hydrophilic-Interaction/Anion-Exchange Liquid Chromatography Mass Spectrometry (Unified-HILIC/AEX/MS): A Single-Run Method for Comprehensive and Simultaneous Analysis of Polar Metabolome.

Kohta Nakatani, Yoshihiro Izumi, Masatomo Takahashi, Takeshi Bamba.

One of the technical challenges in the field of metabolomics is the development of a single-run method to detect the full complement of polar metabolites in biological samples. However, an ideal method to meet this demand has not yet been developed. Herein, we proposed a simple methodology that enables the comprehensive and simultaneous analysis of polar metabolites using unified-hydrophilic-interaction/anion-exchange liquid chromatography mass spectrometry (unified-HILIC/AEX/MS) with a polymer-based mixed amines column composed of methacrylate-based polymer particles with primary, secondary, tertiary, and quaternary amines as functional groups. The optimized unified-HILIC/AEX/MS method is composed of two consecutive chromatographic separations, HILIC-dominant separation for cationic, uncharged, and zwitterionic polar metabolites [retention times (RTs) = 0-12.8 min] and AEX-dominant separation for polar anionic metabolites (RTs = 12.8-26.5 min), by varying the ratio of acetonitrile to 40 mM ammonium bicarbonate solution (pH 9.8). A total of 400 polar metabolites were analyzed simultaneously through a combination of highly efficient separation using unified-HILIC/AEX and remarkably sensitive detection using multiple reaction monitoring-based triple quadrupole mass spectrometry (unified-HILIC/AEX/MS/MS). A nontargeted metabolomic approach using unified-HILIC/AEX high-resolution mass spectrometry (unified-HILIC/AEX/HRMS) also provided more comprehensive information on polar metabolites (3242 metabolic features) in HeLa cell extracts than the conventional HILIC/HRMS method (2068 metabolic features). Our established unified-HILIC/AEX/MS/MS and unified-HILIC/AEX/HRMS methods have several advantages over conventional techniques, including polar metabolome coverage, throughput, and accurate quantitative performance, and represent potentially useful tools for in-depth studies on metabolism and biomarker discovery.

DOI: https://doi.org/10.1021/acs.analchem.2c03986
Toxics. 2022 Nov 12. pii: 687. [Epub ahead of print]10(11):

Determination of 19 Steroid Hormones in Human Serum and Urine Using Liquid Chromatography-Tandem Mass Spectrometry.

Zhong-Min Li, Kurunthachalam Kannan.

  This paper describes a methodology for simultaneous determination of 19 steroid hormones, viz. estrone, estradiol, estriol, testosterone, 5α-dihydrotestosterone, androstenedione, androstenediol, dehydroepiandrosterone, progesterone, pregnenolone, 17α-OH-progesterone, 17α-OH-pregnenolone, cortisone, cortisol, 11-deoxycortisol, 11-deoxycorticosterone, 11-dehydrocorticosterone, aldosterone, and corticosterone, in 500-µL of urine or serum/plasma. The method was optimized using isotopically labeled internal standards and liquid-liquid extraction followed by detection using liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-MS/MS). Dansylation of estrogens significantly improved their sensitivities (~11- to 23-fold) and chromatographic separation. The respective limit of detection (LOD) and limit of quantification (LOQ) of all analytes were 0.04-0.28 and 0.14-0.92 ng/mL in human urine, and 0.11-0.35 and 0.38-1.18 ng/mL in human serum/plasma. Recoveries of all analytes (except for progesterone) fortified at 10, 20, and 200 ng/mL in urine and serum were 80-120%, with standard deviations ranging from 0 to 17.3%. Repeated analysis of similarly fortified urine and serum samples yielded intra-day and inter-day variations of 0-21.7% and 0.16-11.5%, respectively. All analytes except cortisone exhibited weak matrix effects in urine and serum (-13.9-18.2%). The method was further validated through the analysis of the National Institute of Standards and Technology (NIST) plasma Standard Reference Material (SRM1950) with certified concentrations for cortisol, progesterone, and testosterone (coefficient of variation: 3-11%). The developed method was applied in the analysis of urine samples from 20 volunteers, which revealed the occurrence of 16 analytes with detection frequencies (DFs) > 80%. Furthermore, 15 analytes were found in plasma SRM1950, indicating the feasibility of our method in the analysis of steroid hormones in urine and serum/plasma. This method will facilitate analysis of steroid hormones in population-based biomonitoring studies.

Keywords:  androgens; corticosteroids; estrogens; progestogens; serum; steroid hormones; urine

DOI:  https://doi.org/10.3390/toxics10110687
J Pharm Biomed Anal. 2022 Nov 14. pii: S0731-7085(22)00583-0. [Epub ahead of print]224 115162

Branched medium-chain fatty acid profiling and enantiomer separation of anteiso-forms of teicoplanin fatty acyl side chain RS3 using UHPLC-MS/MS with polysaccharide columns.

Christian Geibel, Matthias Olfert, Cornelius Knappe, Kristian Serafimov, Michael Lämmerhofer.

  This work reports on targeted UHPLC-tandem mass spectrometry methods for the chiral separation of anteiso-methyl branched fatty acids (aiFAs). The methods involve precolumn derivatization with 1-naphthylamine and chiral separation on Chiralpak IG-U. anteiso-Methyl branched fatty acids with up to eight carbons can be separated. A method was used for the assignment of the absolute configuration of an aiFA present as fatty acyl residue of the teicoplanin mixture, namely teicoplanin RS3. Furthermore, the excellent methylene selectivity and improved selectivity for constitutional isomers of the polysaccharide columns was exploited for the elucidation and structural confirmation of previously unknown fatty acyl residues in teicoplanin. This shows the versatility and practical applicability of polysaccharide columns as orthogonal stationary phases to reversed-phase for structural elucidation of natural compounds. The developed methods are useful tools for related subdisciplines such as targeted metabolomics and lipidomics.

Keywords:  Antibiotics; Chiral column; Polysaccharide; Structure elucidation; Targeted lipidomics; Teicoplanin

DOI:  https://doi.org/10.1016/j.jpba.2022.115162
J Pharm Biomed Anal. 2023 Jan 20. pii: S0731-7085(22)00569-6. [Epub ahead of print]223 115148

A sensitive and accurate GC-MS method for analyzing microbial metabolites short chain fatty acids and their hydroxylated derivatives in newborn fecal samples.

Fangbo Xia, Lishan Guo, Peng Cui, Qian Xu, Junda Huang, Hongwei Zhou, Wei Shen.

  Short chain fatty acids (SCFAs), crucial intestinal bacterial metabolites, have been widely accepted as potential diagnostic markers in neonatal medicine. Nevertheless, it is still a great challenge to accurately quantify SCFAs in newborn fecal samples due to the huge variation of water content, limited commercial isotope-labeled internal standards and poor sensitivity. In this study, Na2CO3 solution (50 μg/mL) was applied to convert the free SCFAs to SCFA sodium salts, which could prevent the loss of violate SCFAs during lyophilization process. Furthermore, N-methylbenzylamine-d0/d3 was applied as the chemical derivatization regent to enhance the sensitivity and accuracy. Based on this method, the SCFA contents in meconium and neonatal fecal samples were analyzed to illustrate the change of SCFAs during the gut microbiome development. Chemical derivatization based on N-methylbenzylamine-d0/d3 could not only significantly promote the sensitivity (323-1280 folds compared to free SCFAs) by promoting the ionization efficiency, but also provide one-to-one isotope internal standards. Moreover, 7 SCFAs, including acetic acid (2), n-butyric acid (4), isobutyric acid (5), 2-hydroxybutyric acid (11), 2-hydroxy-3-methylbutyric acid (13), 3-hydroxybutyric acid (14), 2-hydroxy-2-methylbutyric acid (17) were found to be significantly increased in neonatal fecal samples compared to the meconium fecal samples. All these results proved that this method could be applied for SCFA analysis in newborn fecal samples with perfect accuracy and sensitivity.

Keywords:  Chemical derivatization; Gas chromatography-mass spectrometry; Newborn fecal sample; Short chain fatty acids

DOI:  https://doi.org/10.1016/j.jpba.2022.115148
Metabolites. 2022 Nov 15. pii: 1111. [Epub ahead of print]12(11):

Lipid Serum Profiling of Boar-Tainted and Untainted Pigs Using GC×GC-TOFMS: An Exploratory Study.

Kinjal Bhatt, Thibaut Dejong, Lena M Dubois, Alice Markey, Nicolas Gengler, José Wavreille, Pierre-Hugues Stefanuto, Jean-François Focant.

  Mass spectrometry (MS)-based techniques, including liquid chromatography coupling, shotgun lipidomics, MS imaging, and ion mobility, are widely used to analyze lipids. However, with enhanced separation capacity and an optimized chemical derivatization approach, comprehensive two-dimensional gas chromatography (GC×GC) can be a powerful tool to investigate some groups of small lipids in the framework of lipidomics. This study describes the optimization of a dedicated two-stage derivatization and extraction process to analyze different saturated and unsaturated fatty acids in plasma by two-dimensional gas chromatography-time-of-flight mass spectrometry (GC×GC-TOFMS) using a full factorial design. The optimized condition has a composite desirability of 0.9159. This optimized sample preparation and chromatographic condition were implemented to differentiate between positive (BT) and negative (UT) boar-tainted pigs based on fatty acid profiling in pig serum using GC×GC-TOFMS. A chemometric screening, including unsupervised (PCA, HCA) and supervised analysis (PLS-DA), as well as univariate analysis (volcano plot), was performed. The results suggested that the concentration of PUFA ω-6 and cholesterol derivatives were significantly increased in BT pigs, whereas SFA and PUFA ω-3 concentrations were increased in UT pigs. The metabolic pathway and quantitative enrichment analysis suggest the significant involvement of linolenic acid metabolism.

Keywords:  GC×GC–TOFMS; boar taint; fatty acids; gas chromatography; lipidomics

DOI:  https://doi.org/10.3390/metabo12111111
Metabolites. 2022 Nov 14. pii: 1110. [Epub ahead of print]12(11):

An Optimised Monophasic Faecal Extraction Method for LC-MS Analysis and Its Application in Gastrointestinal Disease.

Patricia E Kelly, H Jene Ng, Gillian Farrell, Shona McKirdy, Richard K Russell, Richard Hansen, Zahra Rattray, Konstantinos Gerasimidis, Nicholas J W Rattray.

  Liquid chromatography coupled with mass spectrometry (LC-MS) metabolomic approaches are widely used to investigate underlying pathogenesis of gastrointestinal disease and mechanism of action of treatments. However, there is an unmet requirement to assess faecal metabolite extraction methods for large-scale metabolomics studies. Current methods often rely on biphasic extractions using harmful halogenated solvents, making automation and large-scale studies challenging. The present study reports an optimised monophasic faecal extraction protocol that is suitable for untargeted and targeted LC-MS analyses. The impact of several experimental parameters, including sample weight, extraction solvent, cellular disruption method, and sample-to-solvent ratio, were investigated. It is suggested that a 50 mg freeze-dried faecal sample should be used in a methanol extraction (1:20) using bead beating as the means of cell disruption. This is revealed by a significant increase in number of metabolites detected, improved signal intensity, and wide metabolic coverage given by each of the above extraction parameters. Finally, we addressed the applicability of the method on faecal samples from patients with Crohn's disease (CD) and coeliac disease (CoD), two distinct chronic gastrointestinal diseases involving metabolic perturbations. Untargeted and targeted metabolomic analysis demonstrated the ability of the developed method to detect and stratify metabolites extracted from patient groups and healthy controls (HC), highlighting characteristic changes in the faecal metabolome according to disease. The method developed is, therefore, suitable for the analysis of patients with gastrointestinal disease and can be used to detect and distinguish differences in the metabolomes of CD, CoD, and HC.

Keywords:  Crohn’s disease; coeliac disease; inflammatory bowel disease; mass spectrometry; metabolite extraction

DOI:  https://doi.org/10.3390/metabo12111110
Molecules. 2022 Nov 16. pii: 7945. [Epub ahead of print]27(22):

The Development of New Methodology for Determination of Vincristine (VCR) in Human Serum Using LC-MS/MS-Based Method for Medical Diagnostics.

Kamil Jurowski, Łukasz Paprotny, Marcin Zakrzewski, Dorota Wianowska, Joanna Kasprzyk-Pochopień, Małgorzata Herman, Katarzyna Madej, Wojciech Piekoszewski, Tomasz Kubrak.

  In this article, we have presented the development and validation of a rapid and sensitive reversed-phase liquid chromatography with tandem mass spectrometry (LC-MS/MS) method for the determination of vincristine (VCR) in patient serum samples. Chromatographic separation was achieved on a Kinetex® (Singapore) column using a mobile phase consisting of 25 mM acetic acid and 0.3% formic acid (A) and methanol (B) in a gradient elution mode at a flow rate of 0.3 mL/min. The VCR and internal standard (vinblastine) were monitored using the multiple reaction monitoring mode under positive electrospray ionization. The lower limit of quantification (LLOQ) was 0.67 ng/mL, and the upper limit of quantification (ULOQ) was 250 ng/mL for VCR. The calculated values of LOD and LOQ for VCR were 0.075 and 0.228 ng/mL, respectively. The calibration curve was linear over the VCR concentration range of 1.0-250 ng/mL in serum. The intra- and inter-day precision and precision were within the generally accepted criteria for the bioanalytical method (<15%). The method was successfully applied to the analysis of serum samples in clinical practice.

Keywords:  LC-MS/MS method; clinical practice; oncovine; vincristine

DOI:  https://doi.org/10.3390/molecules27227945
Molecules. 2022 Nov 16. pii: 7937. [Epub ahead of print]27(22):

LC-HRMS/MS-Based Metabolomics Approaches Applied to the Detection of Antifungal Compounds and a Metabolic Dynamic Assessment of Orchidaceae.

Gesiane S Lima, Nerilson M Lima, Jussara V Roque, Deborah V A de Aguiar, João V A Oliveira, Gabriel F Dos Santos, Andrea R Chaves, Boniek G Vaz.

  The liquid chromatography-mass spectrometry (LC-MS)-based metabolomics approach is a powerful technology for discovering novel biologically active molecules. In this study, we investigated the metabolic profiling of Orchidaceae species using LC-HRMS/MS data combined with chemometric methods and dereplication tools to discover antifungal compounds. We analyze twenty ethanolic plant extracts from Vanda and Cattleya (Orchidaceae) genera. Molecular networking and chemometric methods were used to discriminate ions that differentiate healthy and fungal-infected plant samples. Fifty-three metabolites were rapidly annotated through spectral library matching and in silico fragmentation tools. The metabolomic profiling showed a large production of polyphenols, including flavonoids, phenolic acids, chromones, stilbenoids, and tannins, which varied in relative abundance across species. Considering the presence and abundance of metabolites in both groups of samples, we can infer that these constituents are associated with biochemical responses to microbial attacks. In addition, we evaluated the metabolic dynamic through the synthesis of stilbenoids in fungal-infected plants. The tricin derivative flavonoid- and the loliolide terpenoidfound only in healthy plant samples, are promising antifungal metabolites. LC-HRMS/MS, combined with state-of-the-art tools, proved to be a rapid and reliable technique for fingerprinting medicinal plants and discovering new hits and leads.

Keywords:  antifungal compounds; liquid chromatography–mass spectrometry; metabolic dynamic; untargeted metabolomics

DOI:  https://doi.org/10.3390/molecules27227937
Front Mol Biosci. 2022 ;9 1049016

Applications of liquid chromatography-mass spectrometry based metabolomics in predictive and personalized medicine.

Juntuo Zhou, Lijun Zhong.

  Metabolomics is a fast-developing technique used in biomedical researches focusing on pathological mechanism illustration or novel biomarker development for diseases. The ability of simultaneously quantifying thousands of metabolites in samples makes metabolomics a promising technique in predictive or personalized medicine-oriented researches and applications. Liquid chromatography-mass spectrometry is the most widely employed analytical strategy for metabolomics. In this current mini-review, we provide a brief update on the recent developments and novel applications of LC-MS based metabolomics in the predictive and personalized medicine sector, such as early diagnosis, molecular phenotyping or prognostic evaluation. COVID-19 related metabolomic studies are also summarized. We also discuss the prospects of metabolomics in precision medicine-oriented researches, as well as critical issues that need to be addressed when employing metabolomic strategy in clinical applications.

Keywords:  COVID; liquid chromatography; mass spectrometry; metabolomics; personalized medicine; predictive medicine

DOI:  https://doi.org/10.3389/fmolb.2022.1049016
J Proteome Res. 2022 Nov 22.

MZA: A Data Conversion Tool to Facilitate Software Development and Artificial Intelligence Research in Multidimensional Mass Spectrometry.

Aivett Bilbao, Dylan H Ross, Joon-Yong Lee, Micah T Donor, Sarah M Williams, Ying Zhu, Yehia M Ibrahim, Richard D Smith, Xueyun Zheng.

  Modern mass spectrometry-based workflows employing hybrid instrumentation and orthogonal separations collect multidimensional data, potentially allowing deeper understanding in omics studies through adoption of artificial intelligence methods. However, the large volume of these rich spectra challenges existing data storage and access technologies, therefore precluding informatics advancements. We present MZA (pronounced m-za), the mass-to-charge (m/z) generic data storage and access tool designed to facilitate software development and artificial intelligence research in multidimensional mass spectrometry measurements. Composed of a data conversion tool and a simple file structure based on the HDF5 format, MZA provides easy, cross-platform and cross-programming language access to raw MS-data, enabling fast development of new tools in data science programming languages such as Python and R. The software executable, example MS-data and example Python and R scripts are freely available at https://github.com/PNNL-m-q/mza.

Keywords:  data conversion; data-independent acquisition; ion mobility spectrometry; mass spectrometry; open data format

DOI:  https://doi.org/10.1021/acs.jproteome.2c00313
J Chromatogr A. 2022 Nov 07. pii: S0021-9673(22)00832-9. [Epub ahead of print]1685 463641

Use of tandem carbon microfiber columns for on-line fractionation strategy of reducing ion suppression effects in electrospray ionization-mass spectrometry.

Zhao Wang, Hai-Bo Shang, Meiyu Cui, Biao Ma, Donghao Li, Dongri Jin, Meihua Dong.

  As we all know, the complexity and diversity of complex sample are confronting with challenge of high-sensitive mass spectrometry analysis, especially direct mass spectrometry. The work proposed a two-dimensional carbon microfiber fractionation (2DμCFs) system for the reduction of ion suppression effects in electrospray ionization mass spectrometry (ESI-MS). The 2DμCFs system can on-line fractionated the complex sample into strong-polar, medium-polar and weak-polar fractions for sequential MS analysis. Direct analysis brings about the strong ion suppression effect up to 85%, but the fractionated analysis of 2DμCFs system can distinctly reduce the ion suppression effect to less than 43%, even close to none. And the fractionated analysis not only decrease the number of analytes of direct analysis, but also narrows down the polarity range of analytes within the droplets of ESI, contributing to the homogeneous distribution to reduce the ion suppression effect. As an example, the 2DμCFs system coupled with tandem mass spectrometry (MS/MS) was applied for fractionated analysis of Radix Puerariae extract in 4.5 min. Compared with direct MS/MS, the 2DμCFs-MS/MS shows the lower ion suppression and the more ionic species (m/z). In addition, and most of ionic species detected by the 2DμCFs-MS/MS, are the same as those by HPLC MS/MS. Furthermore, the 2DμCFs-MS/MS exhibit the good analysis repeatability of real sample with the RSDs less than 10.32% (intra-day), 7.12% (inter-day) and 14.28% (inter-batch of CFs and ACFs). The carbon fibers (CFs) and active carbon fibers (ACFs) columns, as the key parts, are conducive to achieve on-line fractionation of compounds based on the difference of polarity. The 2DμCFs system has the merits of on-line, speediness, low-pressure and recycle. More importantly, such fast and high-throughput method is advantageous for comprehensive screening of complex samples in drug, clinical, environment and plant.

Keywords:  Carbon fiber; ESI-MS; Fractionated analysis; Ion suppression effects; Radix Puerariae

DOI:  https://doi.org/10.1016/j.chroma.2022.463641
Bioengineering (Basel). 2022 Nov 18. pii: 707. [Epub ahead of print]9(11):

The Exploration of Microbial Natural Products and Metabolic Interaction Guided by Mass Spectrometry Imaging.

Hao Li, Zhiyong Li.

  As an impressive mass spectrometry technology, mass spectrometric imaging (MSI) can provide mass spectra data and spatial distribution of analytes simultaneously. MSI has been widely used in diverse fields such as clinical diagnosis, the pharmaceutical industry and environmental study due to its accuracy, high resolution and developing reproducibility. Natural products (NPs) have been a critical source of leading drugs; almost half of marketed drugs are derived from NPs or their derivatives. The continuous search for bioactive NPs from microorganisms or microbiomes has always been attractive. MSI allows us to analyze and characterize NPs directly in monocultured microorganisms or a microbial community. In this review, we briefly introduce current mainstream ionization technologies for microbial samples and the key issue of sample preparation, and then summarize some applications of MSI in the exploration of microbial NPs and metabolic interaction, especially NPs from marine microbes. Additionally, remaining challenges and future prospects are discussed.

Keywords:  mass spectrometry imaging; metabolic interaction; microorganism; natural products

DOI:  https://doi.org/10.3390/bioengineering9110707
Rapid Commun Mass Spectrom. 2022 Nov 21. e9437

How searching against multiple libraries can lead to biased results in GC/MS-based metabolomics.

Andrey S Samokhin, Dmitriy D Matyushin.

RATIONALE: Databases of electron ionization mass spectra are often used in GC/MS-based untargeted metabolomics analysis. The results of the library search depend on several factors such as the size and quality of the database, the library search algorithm, and etc. We found out that the list of considered m/z values is another important parameter. Unfortunately, this information is not usually specified by software developers, and it is hidden from the end user.METHODS: We created synthetic data sets and figured out how several popular software products (AMDIS, ChromaTOF, MS Search, and Xcalibur) select the list of m/z values for the library search. Moreover, we considered data sets of real mass spectra (presented in both NIST and FiehnLib libraries) and compared the library search results obtained within different software products. All programs under consideration call the NIST MS Search binaries to perform the library search using the Identity algorithm.
RESULTS: We found out that AMDIS and ChromaTOF can give biased library search results under particular conditions. In untargeted metabolomics, it can happen when NIST and FiehnLib libraries are used simultaneously, the scan range of the instrument is less than 85, and the correct answer is present only in the FiehnLib library.
CONCLUSIONS: The main reason for biased results is that the information about the scan range is not stored in the metadata of library records. As a result, in the case of AMDIS and ChromaTOF software, some unrecorded peaks are considered as missing ones during the library search, the respective compound is penalized, and the correct answer falls outside the top 5 or even 10 hits. At the same time, the default algorithm for selecting the list of considered m/z values implemented in MS Search is free from such unexpected behavior.

DOI: https://doi.org/10.1002/rcm.9437
Rapid Commun Mass Spectrom. 2022 Nov 22. e9439

High-Resolution Ion Mobility Spectrometry Mass Spectrometry for Isomeric Separation of Prostanoids after Girard's reagent T Derivatization.

L Lamont, D Hadavi, A P Bowman, B Flinders, D Cooper-Shepherd, M Palmer, J Jordens, Y Mengerink, M Honing, J Langridge, T Porta Siegel, R J Vreeken, R M A Heeren.

RATIONALE: Isomeric separation of prostanoids is often a challenge and requires chromatography and time-consuming sample preparation. Multiple prostanoid isomers have distinct in vivo functions crucial to understand the inflammation process, including prostaglandins E2 (PGE2 ) and D2 (PGD2 ). High-resolution Ion Mobility Spectrometry (IMS) based on linear ion transport in low-to-moderate electric fields and nonlinear ion transport in strong electric fields emerges as a broad approach for rapid separations prior to mass spectrometry (MS).METHODS: Derivatization with Girard's reagent T (GT) was used to overcome inefficient ionization of prostanoids in negative ionization mode due to poor deprotonation of the carboxylic acid group. Three high-resolution IMS techniques including linear cyclic IMS (cIMS), linear Trapped IMS (TIMS), and nonlinear high-Field Asymmetric Waveform IMS (FAIMS) were compared for the isomeric separation and endogenous detection of prostanoids present in intestinal tissue.
RESULTS: Direct infusion of GT derivatized prostanoids proved to increase the ionization efficiency in positive ionization mode by a factor of >10, which enabled detection of these molecules in endogenous concentration levels. The high-resolution IMS comparison revealed its potential for rapid isomeric analysis of biologically relevant prostanoids. Strengths and weaknesses of both linear and nonlinear IMS were discussed. Endogenous prostanoid detection in intestinal tissue extracts demonstrated the applicability of our approach in biomedical research.
CONCLUSIONS: The applied derivatization strategy offers high sensitivity and improved stereoisomeric separation for screening of complex biological systems. The high-resolution IMS comparison indicated that the best sensitivity and resolution are achieved by linear and nonlinear IMS, respectively.

DOI: https://doi.org/10.1002/rcm.9439
Toxics. 2022 Nov 14. pii: 688. [Epub ahead of print]10(11):

Evaluating Different Extraction Approaches for GC-MS Based Metabolomics Analysis of the Giant Pandas' Fur.

Yang Yang, Yanqiang Yin, Xianglan Tang, Yinyin Xia, Jinya Zhang, Chun Yan, Weixuan Zhang, Hua Zhang, Ting-Li Han.

  Giant pandas in zoo captivity are situated in residential areas, where environmental pollutants and anthropogenic factors have an impact on their health. Hair metabolomics has been applied in numerous environmental toxicological studies. Therefore, the panda fur metabolome could be a reliable approach to reflect endogenous and exogenous metabolic changes related to environmental exposure. However, there is no established extraction protocol to study the fur metabolome of pandas. The aim of this research was to optimize the extraction of panda fur metabolome for high-throughput metabolomics analysis using gas chromatography-mass spectrometry. Fur samples were collected from five pandas. Eight different extraction methods were investigated and evaluated for their reproducibility, metabolite coverage, and extraction efficiency, particularly in relation to the biochemical compound classes such as amino acids, tricarboxylic acid cycle derivatives, fatty acids, and secondary metabolites. Our results demonstrated that HCl + ACN were the superior extraction solvents for amino acid and secondary metabolite extraction, and NaOH + MeOH was ideal for fatty acid extraction. Interestingly, the metabolomic analysis of panda fur was capable of discriminating the longitudinal metabolite profile between black and white furs. These extraction protocols can be used in future study protocols for the analysis of the fur metabolome in pandas.

Keywords:  GC-MS; endogenous and exogenous metabolome; extraction method; giant pandas

DOI:  https://doi.org/10.3390/toxics10110688
Int J Mol Sci. 2022 Nov 10. pii: 13858. [Epub ahead of print]23(22):

Comprehensive Steroid Assay with Non-Targeted Analysis Using Liquid Chromatography Ion Mobility Mass Spectrometry.

Mai Yamakawa, Shigehiro Karashima, Riko Takata, Taichi Haba, Keigo Kuroiwa, Hideaki Touyama, Atsushi Hashimoto, Seigo Konishi, Daisuke Aono, Mitsuhiro Kometani, Hidetaka Nambo, Takashi Yoneda, Issey Osaka.

  Aldosterone-producing adenomas (APAs) have different steroid profiles in serum, depending on the causative genetic mutation. Ion mobility is a separation technique for gas-phase ions based on their m/z values, shapes, and sizes. Human serum (100 µL) was purified by liquid-liquid extraction using tert-butyl methyl ether/ethyl acetate at 1/1 (v/v) and mixed with deuterium-labeled steroids as the internal standard. The separated supernatant was dried, re-dissolved in water containing 20% methanol, and injected into a liquid chromatography-ion mobility-mass spectrometer (LC/IM/MS). We established a highly sensitive assay system by separating 20 steroids based on their retention time, m/z value, and drift time. Twenty steroids were measured in the serum of patients with primary aldosteronism, essential hypertension, and healthy subjects and were clearly classified using principal component analysis. This method was also able to detect phosphatidylcholine and phosphatidylethanolamine, which were not targeted. LC/IM/MS has a high selectivity for known compounds and has the potential to provide information on unknown compounds. This analytical method has the potential to elucidate the pathogenesis of APA and identify unknown steroids that could serve as biomarkers for APA with different genetic mutations.

Keywords:  LC/IM/MS; aldosterone; aldosterone-related disorder; ion mobility; non-target; quantitation; steroid

DOI:  https://doi.org/10.3390/ijms232213858
Foods. 2022 Nov 13. pii: 3624. [Epub ahead of print]11(22):

Simultaneous Determination of 11 Mycotoxins in Maize via Multiple-Impurity Adsorption Combined with Liquid Chromatography-Tandem Mass Spectrometry.

Xin Guan, Yuchao Feng, Decheng Suo, Zhiming Xiao, Shi Wang, Ying Liang, Xia Fan.

  In this study, multiple-impurity adsorption purification (MIA) technologies and liquid chromatography-tandem mass spectrometry (LC-MS/MS) were used to establish a method for detecting 11 mycotoxins in maize. The conditions for mass spectrometry and MIA were optimized. Maize was extracted with 70% acetonitrile solution, enriched, and purified using MIA technologies, and then, analyzed via LC-MS/MS. The results showed that the linear correlation coefficients of the 11 mycotoxins were >0.99, the sample recoveries ranged from 77.5% to 98.4%, and the relative standard deviations were <15%. The validated method was applied to investigate actual samples, and the results showed that the main contaminating toxins in maize were aflatoxins (AFs), deoxynivalenol (DON), fumonisins (FBs), ochratoxin A (OTA), and zearalenone (ZEN). Additionally, simultaneous contamination by multiple toxins was common. The maximum detection values of the mycotoxins were 77.65, 1280.18, 200,212.41, 9.67, and 526.37 μg/kg for AFs, DON, FBs, OTA, and ZEN, respectively. The method is simple in pre-treatment, convenient in operation, and suitable for the simultaneous determination of 11 types of mycotoxins in maize.

Keywords:  liquid chromatography–tandem mass spectrometry; multiple-impurity adsorption; mycotoxins; simultaneous detection

DOI:  https://doi.org/10.3390/foods11223624
Drug Discov Today. 2022 Nov 22. pii: S1359-6446(22)00453-6. [Epub ahead of print] 103460

Metabolomics- and systems-biology-guided discovery of metabolite lead compounds and druggable targets.

Amelia Palermo.

  Metabolomics enables the comprehensive and unbiased analysis of metabolites and lipids in biological systems. In conjunction with high-throughput activity screening, big data and synthetic biology, metabolomics can guide the discovery of lead compounds with pharmacological activity from natural sources and the gut microbiome. In combination with other omics, metabolomics can further unlock the elucidation of compound toxicity, the mode of action and novel druggable targets of disease. Here, we discuss the workflows, limitations and future opportunities to leverage metabolomics and big data in conjunction with systems and synthetic biology for streamlining the discovery and development of molecules of pharmaceutical interest.

Keywords:  Metabolomics; artificial intelligence; big data; drug discovery; drug target; mode of action; synthetic biology; systems biology

DOI:  https://doi.org/10.1016/j.drudis.2022.103460
Molecules. 2022 Nov 20. pii: 8058. [Epub ahead of print]27(22):

High-Throughput Metabolic Soft-Spot Identification in Liver Microsomes by LC/UV/MS: Application of a Single Variable Incubation Time Approach.

Yanlin Zhu, Guiying Chen, Kerong Zhang, Chang Chen, Weiqing Chen, Mingshe Zhu, Hongliang Jiang.

  CYP-mediated fast metabolism may lead to poor bioavailability, fast drug clearance and significant drug interaction. Thus, metabolic stability screening in human liver microsomes (HLM) followed by metabolic soft-spot identification (MSSID) is routinely conducted in drug discovery. Liver microsomal incubations of testing compounds with fixed single or multiple incubation time(s) and quantitative and qualitative analysis of metabolites using high-resolution mass spectrometry are routinely employed in MSSID assays. The major objective of this study was to develop and validate a simple, effective, and high-throughput assay for determining metabolic soft-spots of testing compounds in liver microsomes using a single variable incubation time and LC/UV/MS. Model compounds (verapamil, dextromethorphan, buspirone, mirtazapine, saquinavir, midazolam, amodiaquine) were incubated at 3 or 5 µM with HLM for a single variable incubation time between 1 and 60 min based on predetermined metabolic stability data. As a result, disappearances of the parents were around 20-40%, and only one or a few primary metabolites were generated as major metabolite(s) without notable formation of secondary metabolites. The unique metabolite profiles generated from the optimal incubation conditions enabled LC/UV to perform direct quantitative estimation for identifying major metabolites. Consequently, structural characterization by LC/MS focused on one or a few major primary metabolite(s) rather than many metabolites including secondary metabolites. Furthermore, generic data-dependent acquisition methods were utilized to enable Q-TOF and Qtrap to continuously record full MS and MS/MS spectral data of major metabolites for post-acquisition data-mining and interpretation. Results from analyzing metabolic soft-spots of the seven model compounds demonstrated that the novel MSSID assay can substantially simplify metabolic soft-spot identification and is well suited for high-throughput analysis in lead optimization.

Keywords:  LC/UV/MS; Qtrap mass spectrometry; metabolic soft-spot; metabolite identification; triple-TOF mass spectrometry

DOI:  https://doi.org/10.3390/molecules27228058
J Magn Reson. 2022 Nov 13. pii: S1090-7807(22)00193-8. [Epub ahead of print]345 107335

Chemical shift variations in common metabolites.

Fatema Bhinderwala, Heidi E Roth, Hannah Noel, Dennis Feng, Robert Powers.

  The reliability and robustness of metabolite assignments in 1H NMR is complicated by numerous factors including variations in temperature, pH, buffer choice, ionic strength, and mixture composition that led to peak overlap and spectral crowding. As sample conditions fluctuate, peak drift and line broadening further complicate peak deconvolution and subsequent chemical assignment. We present a collection of 1D 1H NMR spectra of 54 common metabolites at varied pH (6.0 to 8.0 in 0.5 step increments) and temperature (290 K to 308 K) to quantify chemical shift variability to facilitate automated metabolite assignments. Our results illustrate the fundamental challenges with accurately assigning NMR peaks under varied environmental conditions prevalent in complex mixtures. Phosphorylated metabolites showed a larger variation in chemical shifts due to pH, whereas; amino acids showed a higher variation due to temperature. Mixtures of phosphorous compounds showed a consistently poor reliability in achieving an accurate assignment. Phosphorylated cholines, amino acids, and glycerols yielded a 40 % false negative rate for 7 out of 9 mixture conditions. Amino acids had a false negative rate of 57 % at 298 K and pH 8. Our results demonstrate that the automated assignments of complex biofluid mixtures require an expert to intervene to confirm the accuracy of metabolite assignments. Our analysis also indicates the need for reference databases to include spectra under a variety of conditions that includes mixtures and a range of pH and temperature to improve the accuracy and reproducibility of metabolite assignments.

Keywords:  (1)H NMR; Automated metabolite assignment; Biofluids; Temperature; pH

DOI:  https://doi.org/10.1016/j.jmr.2022.107335
Bioanalysis. 2022 Sep;14(18): 1213-1227

Development and multicenter validation of an LC-MS-based bioanalytical method for antisense therapeutics.

Yuchen Sun, Shin-Ichiro Nitta, Kosuke Saito, Ryuta Hosogai, Keiko Nakai, Ryoya Goda, Hisao Shimizu, Hisashi Fujita, Masaaki Kakehi, Kazuyuki Murata, Takeru Yamaguchi, Takeshi Okuzono, Shinichi Yamane, Mitsuhiko Kawabata, Takayuki Matsunuma, Kentaro Takahara, Noriko Kato, Masaki Yamada, Tokuyuki Yoshida, Takao Inoue, Yoshiro Saito.

  Background: Many bioanalytical methods for antisense oligonucleotides (ASOs) using LC-MS have been reported. However, no data have been available on the reproducibility and robustness of a single bioanalytical method for ASOs. As such, in the current study, we evaluated the reproducibility and robustness of LC-MS-based bioanalytical methods for ASOs in multiple laboratories. Methods/Results: Seven independent laboratories were included in this study. Mipomersen was measured by ion-pairing LC-MS (IP-LC-MS) as a model ASO using different LC-MS. The validation results of calibration curve, accuracy, precision and selectivity met the criteria of conventional bioanalytical method validation guidelines using LC/GC-MS for drugs in all laboratories. Meanwhile, carryover (>20%) was detected in three laboratories. Conclusion: We first demonstrated the multicenter-validated IP-LC-MS bioanalytical method for ASOs. Our data showed that the method was sensitive, robust and reproducible. However, the occurrence of carryover should be carefully monitored in its future application.

Keywords:  Clarity OTX; LC–MS bioanalysis; antisense oligonucleotide; ion-pairing reagent; multilaboratory validation

DOI:  https://doi.org/10.4155/bio-2022-0126
Front Pharmacol. 2022 ;13 1044234

An LC-MS/MS method for the simultaneous determination of 18 antibacterial drugs in human plasma and its application in therapeutic drug monitoring.

Wei Lu, Meng Pan, Hongqin Ke, Jun Liang, Wenbin Liang, Ping Yu, Penghua Zhang, Qibin Wang.

  Antimicrobial resistance (AMR) is a major threat to global health due to the wide use of antibacterial drugs. Multiple studies show that the pharmacokinetic/pharmacodynamic (PK/PD) studies of antibiotics are an approach to prevent/delay AMR. The pharmacokinetic parameters of antibiotics are the basis of PK/PD studies, and therapeutic drug monitoring (TDM) is the key method to obtain pharmacokinetic information. We developed an ultra-performance liquid chromatography-tandem mass spectrometry to determine 18 antibacterial drugs (piperacillin, cefazolin, cefuroxime, cefoperazone, ceftriaxone, cefepime, aztreonam, meropenem, imipenem, levofloxacin, moxifloxacin, azithromycin, clindamycin, tigecycline, linezolid, vancomycin, voriconazole and caspofungin) in human plasma for practical clinical usage. Samples were prepared using protein precipitation with methanol. Chromatographic separation was accomplished in 6 min on a BEH C18 column (2.1 × 100 mm, 1.7 µm) using a gradient elution of acetonitrile and 0.1% formic acid in water at a flow rate of 0.3 ml/min. The electrospray ionization source interface was operated in the positive and negative ionization modes. Inter- and intra-day precision, accuracy, recovery, matrix effect, and stability were validated according to the Food and Drug Administration guidance. The correlation coefficients of calibration curves were all greater than 0.99. The accuracies of the 18 antibacterial drugs ranged from 89.1% to 112.4%. The intra-day precision of the analytes ranged from 1.4% to 9.3% and the inter-day precision from 2.1% to 7.2%. The matrix effects ranged from 93.1% to 105.8% and the extraction recoveries ranged between 90.1% and 109.2%. The stabilities of the 18 antibacterial drugs in plasma were evaluated by analyzing three different concentrations following storage at three storage conditions. All samples displayed variations less than 15.0%. The validated method was successfully applied to routine clinical TDM for 231 samples.

Keywords:  UPLC-MS; antibacterial drugs; pharmacodynamics; pharmacokinetics; therapeutic drug monitoring

DOI:  https://doi.org/10.3389/fphar.2022.1044234
J Pharm Biomed Anal. 2023 Jan 20. pii: S0731-7085(22)00584-2. [Epub ahead of print]223 115163

Development of simple and rapid method for Emicizumab quantification by LC-MS/MS in human plasma.

Laurie Josset, Sophie Hodin, Sandrine Delinger, Clémence Marin, Yesim Dargaud, Xavier Delavenne.

  Emicizumab is a new therapeutic monoclonal antibody indicated for prophylaxis in severe haemophilia A patients. Pharmacokinetic variability has been reported in clinical studies, thus dose optimisation based on quantification of plasma drug concentration could be considered to reduce this variability. Therefore, a reliable and accurate quantification of emicizumab is required. In this study, we developed a method for absolute quantification of emicizumab using liquid chromatography coupled to triple quadrupole mass spectrometry (LC-MS/MS). Sample preparation was based on organic solvent precipitation of proteins followed by trypsin digestion. A signature peptide of emicizumab was used for quantification by LC-MS/MS. A stable isotope-labelled peptide was used as an internal standard. Finally, 6 samples from patients treated with emicizumab were quantified by LC-MS/MS and compared with those obtained with the modified one-stage activated partial prothrombin time technique (aPTT) based FVIII activity. The LC-MS/MS method was validated according to FDA recommendations. Good linearity of the calibration curves was observed over the range 5-150 µg/mL. The cross-validation showed an acceptable correlation of the developed LC-MS/MS method with the modified aPTT-based FVIII activity assay, and the Bland-Altman analysis did not show any significant bias.

Keywords:  Emicizumab; Mass spectrometry; Pharmacokinetic; Therapeutic drug monitoring

DOI:  https://doi.org/10.1016/j.jpba.2022.115163
Electrophoresis. 2022 Nov 23.

Ganglioside analysis in body fluids by liquid-phase separation techniques hyphenated to mass spectrometry.

Anca Suteanu-Simulescu, Mirela Sarbu, Raluca Ica, Ligia Petrica, Alina Diana Zamfir.

  The expression of gangliosides in central nervous system (CNS) is a few times higher than in the extraneural tissue, a characteristic highlighting their major role at this level. Although in very low amounts, gangliosides are ubiquitously distributed in body fluids too, where, depending on many factors, including pathological states, their composition fluctuates, thus having diagnostic value. Ganglioside investigation in biological fluids, which, except for cerebrospinal fluid (CSF), may be sampled noninvasively, was for years impeded by the limited sensitivity of the analytical instrumentation available in glycomics. However, since the last decade has witnessed significant developments in biological mass spectrometry (MS) and the hyphenated separation techniques, marked by a major increase in sensitivity, reproducibility and data reliability, ganglioside research started to be focused on biofluid analysis by separation techniques coupled to MS. In this context, our review presents the achievements in this emerging field of gangliosidomics, with a particular emphasis on modern liquid chromatography, thin-layer chromatography, hydrophilic interaction liquid chromatography and ion mobility separation coupled to high performance MS, as well as the results generated by these systems and allied experimental procedures in profiling and structural analysis of gangliosides in healthy or diseased body fluids such as CSF, plasma/serum and milk. This article is protected by copyright. All rights reserved.

Keywords:  gangliosides of body fluids; liquid-phase separation; mass spectrometry; profiling; structural analysis

DOI:  https://doi.org/10.1002/elps.202200229
Biomed Chromatogr. 2022 Nov 21. e5549

Degradation Pathways and Impurity Profiling of Anti-Cancer Drug Apalutamide by HPLC and LC-MS/MS and Separation of Impurities using Design of Experiments.

Narasimha Swamy Lakka, Chandrasekar Kuppan, Niroja Vadagam, S Y Reddamoni, M Chandru.

  Apalutamide an androgen receptor inhibitor is used to treat prostate-cancer. A stability-indicating high-performance liquid chromatography method was developed for assay and organic impurities of Apalutamide in drug substance and in tablet dosages using Design of Experiments. The chromatographic separation was achieved within 30-min using Atlantis dC18 , 100 × 4.6 mm, 3.0-μm and binary gradient program (10-mM KH2 PO4 , pH 3.5; acetonitrile). The detection wavelength, flow rate, column temperature and injection-volume used were 270-nm, 1.0-mL/min, 45°C and 10-μL, respectively. The interaction of independent variables (pH; column temperature; flow rate) and their influences on HPLC parameters were studied using a central composite design, and then the peak separation and elution behaviors between Apalutamide and its seven impurities were determined. The method validation was performed for linearity, detection-limit, quantitation-limit, accuracy, precision and robustness as per ICH. A high-quality recovery with good precision (91.7~106.0%) and correlations (R2 > 0.997) within linear range of 0.12~2.24μg/mL (0.05 ~ 0.3%, w/w) were achieved consistently for assay and organic impurities of Apalutamide. The stability-indicating characteristic of proposed method was assessed through forced degradation and mass balance studies. An effort was made to figure out the chemical structures of newly formed degradation products (DP1 - DP5) using LC-MS/MS.

Keywords:  Apalutamide (ALM); Degradation products; Design of Experiments (DoE); High-performance liquid chromatography (HPLC); Impurity profiling; LC-MS/MS; Stress testing

DOI:  https://doi.org/10.1002/bmc.5549