Journal of Pharmaceutical Analysis

Reducing SARS-CoV-2 pathological protein activity with small molecules

Donata Pluskota-Karwatka, Marcin Hoffmann, Jan Barciszewski

2021, 11(4): 383-397. doi: 10.1016/j.jpha.2021.03.012

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Abstract:
Coronaviruses are dangerous human and animal pathogens. The newly identified coronavirus SARS-CoV-2 is the causative agent of COVID-19 outbreak, which is a real threat to human health and life. The world has been struggling with this epidemic for about a year, yet there are still no targeted drugs and effective treatments are very limited. Due to the long process of developing new drugs, reposition of existing ones is one of the best ways to deal with an epidemic of emergency infectious diseases. Among the existing drugs, there are candidates potentially able to inhibit the SARS-CoV-2 replication, and thus inhibit the infection of the virus. Some therapeutics target several proteins, and many diseases share molecular paths. In such cases, the use of existing pharmaceuticals for more than one purpose can reduce the time needed to design new drugs. The aim of this review was to analyze the key targets of viral infection and potential drugs acting on them, as well as to discuss various strategies and therapeutic approaches, including the possible use of natural products. We highlighted the approach based on increasing the involvement of human deaminases, particularly APOBEC deaminases in editing of SARS-CoV-2 RNA. This can reduce the cytosine content in the viral genome, leading to the loss of its integrity. We also indicated the nucleic acid technologies as potential approaches for COVID-19 treatment. Among numerous promising natural products, we pointed out curcumin and cannabidiol as good candidates for being anti-SARS-CoV-2 agents.

Development of the general chapters of the Chinese Pharmacopoeia 2020 edition: A review

Xinyi Xu, Huayu Xu, Yue Shang, Ran Zhu, Xiaoxu Hong, Zonghua Song, Zhaopeng Yang

2021, 11(4): 398-404. doi: 10.1016/j.jpha.2021.05.001

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Abstract:
The Chinese Pharmacopoeia 2020 edition was reviewed and approved by the National Medical Products Administration and the National Health Commission of the People's Republic of China in July 2020. The current edition was officially implemented on December 30, 2020. The general chapters of the Chinese Pharmacopoeia discuss the general testing methods and guidelines, which are the common requirements and basis for the implementation of drug standards in the Chinese Pharmacopoeia. Owing to adherence to the principles of scientificity, versatility, operability, and sustainable development, there is an improvement in the general chapters of the 2020 edition over those of the previous editions. Further, the application of advanced and mature analytical techniques has expanded, the development of testing methods for exogenous pollutants in traditional Chinese medicines has been strengthened, and technical requirements are now better harmonized with international standards. The updated edition provides technical and methodological support to ensure safety, effectiveness, and control of pharmaceuticals in China and will play an important and active role in encouraging the application of advanced technologies, improving the quality control of medicines, and strengthening the means of drug regulation in China. This review provides a comprehensive introduction of the main features of and changes to the general chapters in the Chinese Pharmacopoeia 2020 edition and aims to provide reference for its correct understanding and accurate implementation.

Liquid chromatographic methods for determination of the new antiepileptic drugs stiripentol, retigabine, rufinamide and perampanel: A comprehensive and critical review

Sara Meirinho, Márcio Rodrigues, Ana Fortuna, Amílcar Falcão, Gilberto Alves

2021, 11(4): 405-421. doi: 10.1016/j.jpha.2020.11.005

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Abstract:
The new antiepileptic drugs perampanel, retigabine, rufinamide and stiripentol have been recently approved for different epilepsy types. Being them an innovation in the antiepileptics armamentarium, a lot of investigations regarding their pharmacological properties are yet to be performed. Besides, considering their broad anticonvulsant activities, an extension of their therapeutic indications may be worthy of investigation, especially regarding other seizure types as well as other central nervous system disorders. Although different liquid chromatographic (LC) methods coupled with ultraviolet, fluorescence, mass or tandem-mass spectrometry detection have already been developed for the determination of perampanel, retigabine, rufinamide and stiripentol, new and more cost-effective methods are yet required. Therefore, this review summarizes the main analytical aspects regarding the liquid chromatographic methods developed for the analysis of perampanel, retigabine (and its main active metabolite), rufinamide and stiripentol in biological samples and pharmaceutical dosage forms. Furthermore, the physicochemical and stability properties of the target compounds will also be addressed. Thus, this review gathers, for the first time, important background information on LC methods that have been developed and applied for the determination of perampanel, retigabine, rufinamide and stiripentol, which should be considered as a starting point if new (bio)analytical techniques are aimed to be implemented for these drugs.

Biocompatible silver nanoparticles: An investigation into their protein binding efficacies, anti-bacterial effects and cell cytotoxicity studies

Sourav Das, Leader Langbang, Mahabul Haque, Vinay Kumar Belwal, Kripamoy Aguan, Atanu Singha Roy

2021, 11(4): 422-434. doi: 10.1016/j.jpha.2020.12.003

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Abstract:
Green synthesis of silver nanoparticles (AgNPs) has garnered tremendous interest as conventional methods include the use and production of toxic chemicals, products, by-products and reagents. In this regard, the synthesis of AgNPs using green tea (GT) extract and two of its components, (−)-epigallocatechin gallate (EGCG) and (+)-catechin (Ct) as capping/stabilizing agents, is reported. The synthesized AgNPs showed antibacterial activity against the bacterial strains Staphylococcus aureus and Escherichia coli, along with anticancer activity against HeLa cells. After administering nanoparticles to the body, they come in contact with proteins and results in the formation of a protein corona; hence we studied the interactions of these biocompatible AgNPs with hen egg white lysozyme (HEWL) as a carrier protein. Static quenching mechanism was accountable for the quenching of HEWL fluorescence by the AgNPs. The binding constant (K_b) was found to be higher for EGCG-AgNPs ((2.309 ± 0.018) × 10⁴ M⁻¹) than for GT-AgNPs and Ct-AgNPs towards HEWL. EGCG-AgNPs increased the polarity near the binding site while Ct-AgNPs caused the opposite effect, but GT-AgNPs had no such observable effects. Circular dichroism studies indicated that the AgNPs had no such appreciable impact on the secondary structure of HEWL. The key findings of this research included the synthesis of AgNPs using GT extract and its constituent polyphenols, and showed significant antibacterial, anticancer and protein-binding properties. The –OH groups of the polyphenols drive the in situ capping/stabilization of the AgNPs during synthesis, which might offer new opportunities having implications for nanomedicine and nanodiagnostics.

Comparative permeability of three saikosaponins and corresponding saikogenins in Caco-2 model by a validated UHPLC-MS/MS method

Siqi Ren, Jingjing Liu, Yunwen Xue, Mei Zhang, Qiwei Liu, Jie Xu, Zunjian Zhang, Rui Song

2021, 11(4): 435-443. doi: 10.1016/j.jpha.2020.06.006

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Abstract:
Saikosaponins (SSs) are the main active components extracted from Bupleuri Radix (BR) which has been used as an important herbal drug in Asian countries for thousands of years. It has been reported that the intestinal bacteria plays an important role in the in vivo disposal of oral SSs. Although the deglycosylated derivatives (saikogenins, SGs) of SSs metabolized by the intestinal bacteria are speculated to be the main components absorbed into the blood after oral administration of SSs, no studies have been reported on the characteristics of SGs for their intestinal absorption, and those for SSs are also limited. Therefore, a rapid UHPLC-MS/MS method was developed to investigate and compare the apparent permeability of three common SSs (SSa, SSd, SSb₂) and their corresponding SGs (SGF, SGG, SGD) through a bidirectional transport experiment on Caco-2 cell monolayer model. The method was validated according to the latest FDA guidelines and applied to quantify the six analytes in transport medium samples extracted via liquid-liquid extraction (LLE). The apparent permeability coefficient (P) determined in this study indicated that the permeability of SGs improved to the moderate class compared to the corresponding parent compounds, predicting a higher in vivo absorption. Moreover, the efflux ratio (ER) value demonstrated an active uptake of SSd and the three SGs, while a passive diffusion of SSa and SSb₂.

Simultaneous determination of fourteen components of Gumiganghwal-tang tablet in human plasma by UPLC-ESI-MS/MS and its application to pharmacokinetic study

Seung-Hyun Jeong, Ji-Hun Jang, Guk-Yeo Lee, Seung-Jung Yang, Hea-Young Cho, Yong-Bok Lee

2021, 11(4): 444-457. doi: 10.1016/j.jpha.2020.08.003

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Abstract:
Gumiganghwal-tang is a traditional herbal medicine widely used for its anti-inflammatory, analgesic, and antipyretic effects. However, the safety and efficacy of its active ingredients based on an in vivo pharmacokinetic (PK) study have yet been investigated. We have established a sensitive and accurate UPLC-ESI-MS/MS method and conducted a PK study on 14 constituents of Gumiganghwal-tang through human plasma analysis. Analytical conditions were optimized according to the physicochemical properties of the 14 compounds to facilitate efficient separation and eliminate overlap or interference between peaks. KINETEX-C₁₈ and Inertsil-C₈ columns were used as UPLC stationary phases, and acetonitrile and aqueous formic acid were used as mobile phases. All the analytes were quantified with a triple quadrupole mass spectrometer using electrospray ionization in multiple reaction monitoring mode. The chromatograms of 14 bioactive compounds showed excellent elution and sensitivity, and each peak was selectively separated and quantified without interference with each other or impurities. The established analytical method was based on international guidelines and was successfully used to perform PK studies of 14 herbal ingredients in humans after oral administration with Gumiganghwal-tang tablets. The oral absorption of most active components of Gumiganghwal-tang was relatively rapid and remained considerably long in the body to be quantified in plasma up to 48 h after administration.

UHPLC-MS/MS analysis of cAMP and cGMP in rat plasma as potential biomarkers of Yin-Yang disharmony in traditional Chinese medicine

Xin Wang, Yue Du, Cuiting Wu, Ming Xu, Youping Liu, Xin Di

2021, 11(4): 458-464. doi: 10.1016/j.jpha.2020.09.001

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Abstract:
Cyclic 3′,5′-adenosine monophosphate (cAMP) and cyclic 3′,5′-guanosine monophosphate (cGMP) are considered as potential biomarkers for Yin-Yang disharmony in traditional Chinese medicine. However, phosphodiesterase-mediated ex vivo degradation of these molecules in biological samples may result in their underestimation. In the present study, a ultra-high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) method was developed for determination of cAMP and cGMP in rat plasma, with special consideration of their stability ex vivo. Following precipitation of proteins from plasma samples with 0.4 M perchloric acid, the analytes were chromatographed on a Shimadzu Shim-pack-XR-ODS II column with 2.5 mM ammonium acetate and methanol in gradient mode. The MS/MS detection was performed using multiple reaction monitoring in the positive electrospray ionization mode. The lower limit of quantification was 0.27 ng/mL for cAMP and 0.37 ng/mL for cGMP. The method was used to determine the plasma cAMP and cGMP levels in normal and Yin deficiency diabetic rats treated with or without Rehmannia glutinosa. The developed method may be useful for evaluating the regulatory effects of Chinese herbal medicine on the levels of cAMP and cGMP in the body.

Determination of bioactive components in the fruits of Cercis chinensis Bunge by HPLC-MS/MS and quality evaluation by principal components and hierarchical cluster analyses

Yuan Hong, Xiaoyan Liao, Zilin Chen

2021, 11(4): 465-471. doi: 10.1016/j.jpha.2020.07.010

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Abstract:
The fruits of leguminous plants Cercis Chinensis Bunge are still overlooked although they have been reported to be antioxidative because of the limited information on the phytochemicals of C. chinensis fruits. A simple, rapid and sensitive HPLC-MS/MS method was developed for the identification and quantitation of the major bioactive components in C. chinensis fruits. Eighteen polyphenols were identified, which are first reported in C. chinensis fruits. Moreover, ten components were simultaneously quantified. The validated quantitative method was proved to be sensitive, reproducible and accurate. Then, it was applied to analyze batches of C. chinensis fruits from different phytomorph and areas. The principal components analysis (PCA) realized visualization and reduction of data set dimension while the hierarchical cluster analysis (HCA) indicated that the content of phenolic acids or all ten components might be used to differentiate C. chinensis fruits of different phytomorph.

Liquid chromatography-mass spectrometry method for the quantification of an anti-sclerostin monoclonal antibody in cynomolgus monkey serum

Yuxiong Gao, Zhendong Chen, Changyong Yang, Dafang Zhong

2021, 11(4): 472-479. doi: 10.1016/j.jpha.2020.08.005

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Abstract:
Liquid chromatography tandem mass spectrometry (LC-MS/MS) has gradually become a promising alternative to ligand binding assay for the bioanalysis of biotherapeutic molecules, due to its rapid method development and high accuracy. In this study, we established a new LC-MS/MS method for the determination of the anti-sclerostin monoclonal antibody (SHR-1222) in cynomolgus monkey serum, and compared it to a previous electrochemiluminescence method. The antibody was quantified by detecting the surrogate peptide obtained by trypsin digestion. The surrogate peptide was carefully selected by investigating its uniqueness, stability and MS response. The quantitative range of the proposed method was 2.00–500 μg/mL, and this verified method was successfully applied to the toxicokinetic assessment of SHR-1222 in cynomolgus monkey serum. It was found that the concentrations of SHR-1222 in cynomolgus monkeys displayed an excellent agreement between the LC-MS/MS and electrochemiluminescence methods (ratios of drug exposure, 0.8–1.0). Notably, two monkeys in the 60 mg/kg dose group had abnormal profiles with a low detection value of SHR-1222 in their individual sample. Combining the high-level anti-drug antibodies (ADAs) in these samples and the consistent quantitative results of the two methods, we found that the decreased concentration of SHR-1222 was due to the accelerated clearance mediated by ADAs rather than the interference of ADAs to the detection platform. Taken together, we successfully developed an accurate, efficient and cost-effective LC-MS/MS method for the quantification of SHR-1222 in serum samples, which could serve as a powerful tool to improve the preclinical development of antibody drugs.

Formation mechanisms of sub-micron pharmaceutical composite particles derived from far- and near-field Raman microscopy

Jakob Hübner, Jean-Baptiste Coty, Yan Busby, Denis Spitzer

2021, 11(4): 480-489. doi: 10.1016/j.jpha.2020.12.002

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Abstract:
Surface enhanced Raman spectroscopy (SERS) and confocal Raman microscopy are applied to investigate the structure and the molecular arrangement of sub-micron furosemide and polyvinylpyrrolidone (furosemide/PVP) particles produced by spray flash evaporation (SFE). Morphology, size and crystallinity of furosemide/PVP particles are analyzed by scanning electron microscopy (SEM) and X-ray powder diffraction (XRPD). Far-field Raman spectra and confocal far-field Raman maps of furosemide/PVP particles are interpreted based on the far-field Raman spectra of pure furosemide and PVP precursors. Confocal far-field Raman microscopy shows that furosemide/PVP particles feature an intermixture of furosemide and PVP molecules at the sub-micron scale. SERS and surface-enhanced confocal Raman microscopy (SECoRM) are performed on furosemide, PVP and furosemide/PVP composite particles sputtered with silver (40 nm). SERS and SECoRM maps reveal that furosemide/PVP particle surfaces mainly consist of PVP molecules. The combination of surface and bulk sensitive analyses reveal that furosemide/PVP sub-micron particles are formed by the agglomeration of primary furosemide nano-crystals embedded in a thin PVP matrix. Interestingly, both far-field Raman microscopy and SECoRM provide molecular information on a statistically-relevant amount of sub-micron particles in a single microscopic map; this combination is thus an effective and time-saving tool for investigating organic sub-micron composites.

Portable and automated analyzer for rapid and high precision in vitro dissolution of drugs

Zhongmei Chi, Siqi Zhao, Xiujun Cui, Yunxiang Feng, Li Yang

2021, 11(4): 490-498. doi: 10.1016/j.jpha.2020.06.001

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Abstract:
We developed a novel portable and automated dissolution test analyzer for rapid and high precision in vitro dissolution testing of drugs. The analyzer consists of a flow-through-cell drug dissolution system, an automated sequential sampling system, a high-speed capillary electrophoresis (HSCE) system, and a data acquisition system. Combining the high-temporal resolution flow-gating sampling approach with HSCE, which has outstanding advantages of efficient separation and resolution, the analyzer can achieve rapid analysis and exhibits the ability in miniaturization for on-site assessment of different active pharmaceutical ingredients. To integrate the flow-through-cell dissolution system with HSCE, a specially designed flow-gating-injection (FGI) interface was employed. The performance of the analyzer was investigated by analyzing the dissolution of immediate-release drugs including single dose (amoxicillin dispersible tablets) and fixed dose combination (amoxicillin and clavulanate potassium) drug tablets with the high-temporal resolutions of 12 s and 20 s, respectively. The dissolution profiles of different active pharmaceutical ingredients could be simultaneously and automatically monitored with high repeatability and accuracy. The analyzer was successfully utilized for the pharmaceutical quality control and bio-relevant dissolution testing, as well as in vivo-in vitro correlation analysis. Our portable analyzer is miniaturized, convenient and of low-cost, and will provide a valuable tool for dissolution testing in pharmaceutical research and development.

Matrix-assisted laser desorption ionization mass spectrometry based quantitative analysis of cordycepin from Cordyceps militaris

Jian Chen, Hai-Fang Li, Guozhu Zhao, Jin-Ming Lin, Xiangwei He

2021, 11(4): 499-504. doi: 10.1016/j.jpha.2021.05.003

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Abstract:
Cordycepin, which has great immunomodulatory activities such as anticancer, antifungal, antivirus, antileukemia and lipid-lowering ones, is the secondary metabolite of Cordyceps militaris (C. militaris). Liquid submerged fermentation is the common cultivation process to produce cordycepin. To optimize the fermentation process and improve production, monitoring the cordycepin secretion in the fermentation is essential. The measurement based on chromatography-mass spectrometry methods is generally involved in the complex sample pretreatments and time-consuming separation, so more rapid and convenient methods are required. Matrix-assisted laser desorption ionization mass spectrometry (MALDI-MS) is more attractive for faster and direct detection. Therefore, MALDI-MS detection combined with isotope-labeled internal standard was applied to the measurement of cordycepin content in the fermentation broth and mycelium. This method made accurate quantification of cordycepin in the range of 5–400 μg/mL with a relative standard deviation of 5.6%. The recovery rates of fermentation samples after the 1, 13, and 25 days were 90.15%, 94.27%, and 95.06%, respectively. The contents of cordycepin in the mycelium and fermentation broth were 136 mg/g and 148.39 mg/mL on the 20th culture day, respectively. The cordycepin secretion curve of the liquid fermentation of C. militaris was real-time traced over 25 days.

Plasma-metabolite-based machine learning is a promising diagnostic approach for esophageal squamous cell carcinoma investigation

Zhongjian Chen, Xiancong Huang, Yun Gao, Su Zeng, Weimin Mao

2021, 11(4): 505-514. doi: 10.1016/j.jpha.2020.11.009

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Abstract:
The aim of this study was to develop a diagnostic strategy for esophageal squamous cell carcinoma (ESCC) that combines plasma metabolomics with machine learning algorithms. Plasma-based untargeted metabolomics analysis was performed with samples derived from 88 ESCC patients and 52 healthy controls. The dataset was split into a training set and a test set. After identification of differential metabolites in training set, single-metabolite-based receiver operating characteristic (ROC) curves and multiple-metabolite-based machine learning models were used to distinguish between ESCC patients and healthy controls. Kaplan-Meier survival analysis and Cox proportional hazards regression analysis were performed to investigate the prognostic significance of the plasma metabolites. Finally, twelve differential plasma metabolites (six up-regulated and six down-regulated) were annotated. The predictive performance of the six most prevalent diagnostic metabolites through the diagnostic models in the test set were as follows: arachidonic acid (accuracy: 0.887), sebacic acid (accuracy: 0.867), indoxyl sulfate (accuracy: 0.850), phosphatidylcholine (PC) (14:0/0:0) (accuracy: 0.825), deoxycholic acid (accuracy: 0.773), and trimethylamine N-oxide (accuracy: 0.653). The prediction accuracies of the machine learning models in the test set were partial least-square (accuracy: 0.947), random forest (accuracy: 0.947), gradient boosting machine (accuracy: 0.960), and support vector machine (accuracy: 0.980). Additionally, survival analysis demonstrated that acetoacetic acid was an unfavorable prognostic factor (hazard ratio (HR): 1.752), while PC (14:0/0:0) (HR: 0.577) was a favorable prognostic factor for ESCC. This study devised an innovative strategy for ESCC diagnosis by combining plasma metabolomics with machine learning algorithms and revealed its potential to become a novel screening test for ESCC.

Visualization analysis of lecithin in drugs based on electrochemiluminescent single gold microbeads

Gen Liu, Pei-Long Wang, Hui Gao

2021, 11(4): 515-522. doi: 10.1016/j.jpha.2021.02.002

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Abstract:
Fast and high-throughput determination of drugs is a key trend in clinical medicine. Single particles have increasingly been adopted in a variety of photoanalytical and electroanalytical applications, and microscopic analysis has been a hot topic in recent years, especially for electrochemiluminescence (ECL). This paper describes a simple ECL method based on single gold microbeads to image lecithin. Lecithin reacts to produce hydrogen peroxide under the successive enzymatic reaction of phospholipase D and choline oxidase. ECL was generated by the electrochemical reaction between a luminol analog and hydrogen peroxide, and ECL signals were imaged by a camera. Despite the heterogeneity of single gold microbeads, their luminescence obeyed statistical regularity. The average luminescence of 30 gold microbeads is correlated with the lecithin concentration, and thus, a visualization method for analyzing lecithin was established. Calibration curves were constructed for ECL intensity and lecithin concentration, achieving detection limits of 0.05 mM lecithin. This ECL imaging platform based on single gold microbeads exhibits outstanding advantages, such as high throughput, versatility and low cost, and holds great potential in disease diagnostics, environmental monitoring and food safety.

A simplified LC-MS/MS method for the quantification of the cardiovascular disease biomarker trimethylamine-N-oxide and its precursors

Katharina Rox, Silke Rath, Dietmar H. Pieper, Marius Vital, Mark Brönstrup

2021, 11(4): 523-528. doi: 10.1016/j.jpha.2021.03.007

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Abstract:
Trimethylamine-N-oxide (TMAO) has emerged as a potential biomarker for atherosclerosis and the development of cardiovascular diseases (CVDs). Although several clinical studies have shown striking associations of TMAO levels with atherosclerosis and CVDs, TMAO determinations are not clinical routine yet. The current methodology relies on isotope-labeled internal standards, which adds to pre-analytical complexity and costs for the quantification of TMAO and its precursors carnitine, betaine or choline. Here, we report a liquid chromatography-tandem mass spectrometry based method that is fast (throughput up to 240 samples/day), consumes low sample volumes (e.g., from a finger prick), and does not require isotope-labeled standards. We circumvented the analytical problem posed by the presence of endogenous TMAO and its precursors in human plasma by using an artificial plasma matrix for calibration. We cross-validated the results obtained using an artificial matrix with those using mouse plasma matrix and demonstrated that TMAO, carnitine, betaine and choline were accurately quantified in ‘real-life’ human plasma samples from healthy volunteers, obtained either from a finger prick or from venous puncture. Additionally, we assessed the stability of samples stored at −20 °C and room temperature. Whereas all metabolites were stable at −20 °C, increasing concentrations of choline were determined when stored at room temperature. Our method will facilitate the establishment of TMAO as a routine clinical biomarker in hematology in order to assess the risk for CVDs development, or to monitor disease progression and intervention effects.

2021 Vol. 11, No. 4