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10 pages, 1648 KiB

Open AccessArticle

Comparison of LC-MS³ and LC-MRM Methods for Quantifying Amantadine and Its Application in Therapeutic Amantadine Monitoring in Human Plasma

by Qiang Sun, Haiwei Cao, Yong Liu, Yanyan Li and Jing Huang

Molecules 2022, 27(21), 7619; https://doi.org/10.3390/molecules27217619 - 07 Nov 2022

Cited by 1 | Viewed by 1542

Abstract

A simple sample preprocessing method was developed for the quantitative determination of amantadine (AMT) in human plasma by liquid chromatography-tandem mass spectrometry cubed (LC-MS³). The LC-MS³ system comprised a Shimadzu Exion LC-20AD HPLC pump coupled with a QTRAP 5500 mass [...] Read more.

A simple sample preprocessing method was developed for the quantitative determination of amantadine (AMT) in human plasma by liquid chromatography-tandem mass spectrometry cubed (LC-MS³). The LC-MS³ system comprised a Shimadzu Exion LC-20AD HPLC pump coupled with a QTRAP 5500 mass spectrometer. First, the plasma samples were pretreated using acetonitrile as the extracting solution to precipitate protein. Next, amantadine and amantadine-d₁₅ (AMT-d₁₅) were separated on an Agilent Poroshell 120 SB-C18 column (4.6 mm × 50 mm, 2.7 μm) using isocratic elution with solvent A (70% 0.1% formic acid) and solvent B (30% acetonitrile) at a flow rate of 0.8 mL/min. The total run time for each sample was 3 min. The system used triple-stage fragmentation transitions at m/z 152.2→135.3→107.4 for AMT quantification in the positive ion mode and m/z 167.0→150.3→118.1 for AMT-d₁₅ quantification. The LC-MS³ assay was linear (r > 0.995) with a concentration range of 50–1500 ng/mL. The lower limit of quantification (LLOQ) was 50 ng/mL, and the intra-day and inter-day accuracies and precisions were less than 8.0% at all concentrations. In addition, the recoveries and matrix effect for AMT in human plasma were within acceptable limits. In terms of stability, AMT had no significant degradation under all conditions. All the results met the requirements of the guidelines of the Food and Drug Administration (FDA) for biological method validation. The novelty of the MS³ assay was that it presented a methodology with higher selectivity and sensitivity. This method was successfully applied to 44 human plasma samples, and the obtained quantitative results were compared with another liquid chromatography-multiple reaction monitoring (LC-MRM) method. The Passing-Bablok regression coefficients and Bland-Altman plot revealed no difference between the LC-MS³ and LC-MRM methods, implying that the developed LC-MS³ method is a reliable and accurate assay for AMT determination in human plasma. These results are also a proof of concept for determining chemicals in biological samples by the LC-MS³ strategy. Full article

(This article belongs to the Special Issue Analytical Applications of Biochemical Mass Spectrometry)

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13 pages, 1968 KiB

Open AccessArticle

Sex Differences of Cardiolipin in Tissue Distribution Based on Targeted Lipidomic Analysis by UHPLC-QTOF-MS/MS

by Yuqi Lin, Xugui Li, Mengxiang Dai, Qiaoyu Li, Qingxin Shi, Lijun Zhang, Rongzeng Huang, Chengwu Song and Shuna Jin

Molecules 2022, 27(20), 6988; https://doi.org/10.3390/molecules27206988 - 18 Oct 2022

Cited by 2 | Viewed by 1647

Abstract

Cardiolipins (CLs) are involved in ATP production, mitochondria biogenesis, apoptosis and mitophagy. Their tissue distribution can provide insight into the function of mitochondria and related diseases. However, the reports on tissue distribution of CLs remain limited. In this research, CLs were identified from [...] Read more.

Cardiolipins (CLs) are involved in ATP production, mitochondria biogenesis, apoptosis and mitophagy. Their tissue distribution can provide insight into the function of mitochondria and related diseases. However, the reports on tissue distribution of CLs remain limited. In this research, CLs were identified from heart, liver, kidney, spleen, lung, skeletal muscle, and brain using ultra-high-performance liquid chromatography coupled with quadrupole time-of-flight mass spectrometry (UHPLC-QTOF-MS/MS). Then, the distribution and sex difference of CLs in seven tissues were compared by a targeted lipidomic approach. A total of 88 CLs were identified, of which 58, 51, 57, 58, 50, 61 and 52 CLs were found in heart, liver, kidney, spleen, lung, skeletal muscle, and brain, respectively. Compared with the distribution of CLs in heart, liver, kidney, and skeletal muscle, the CLs in spleen, lung, and brain showed significant differences. Moreover, the results indicated that there were sex differences of CLs in liver and kidney. A total of 16 CLs in liver tissue and 21 CLs in kidney tissue, with significant sex differences, were screened. Our findings in the targeted lipidomic analysis demonstrated that tissue distribution of CLs was essential in the dynamic states and sex differences of CLs, which might provide evidence for the mitochondrial-related mechanism under physiological and pathological conditions. Full article

(This article belongs to the Special Issue Analytical Applications of Biochemical Mass Spectrometry)

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12 pages, 1947 KiB

Open AccessArticle

An Improved Isotope Labelling Method for Quantifying Deamidated Cobratide Using High-Resolution Quadrupole-Orbitrap Mass Spectrometry

by Bo Liu, Lu Huang, Rongrong Xu, Huihong Fan and Yue Wang

Molecules 2022, 27(19), 6154; https://doi.org/10.3390/molecules27196154 - 20 Sep 2022

Cited by 1 | Viewed by 1192

Abstract

Protein deamidation can severely alter the physicochemical characteristics and biological functions of protein therapeutics. Cobratide is a non-addictive analgesic with wide clinical acceptance. However, the Asn residue at position 48 from the N-terminus of the cobratide amino acid sequence (N48) tends to degrade [...] Read more.

Protein deamidation can severely alter the physicochemical characteristics and biological functions of protein therapeutics. Cobratide is a non-addictive analgesic with wide clinical acceptance. However, the Asn residue at position 48 from the N-terminus of the cobratide amino acid sequence (N48) tends to degrade during purification, storage, and transport. This characteristic could severely affect the drug safety and clinical efficacy of cobratide. Traditional methods for quantitating deamidation reported in previous research are characterised by low efficiency and accuracy; the quality control of cobratide via this method is limited. Herein, we developed an improved ¹⁸O-labelling method based on the detection of a unique peptide (i.e., the protein fragment of cobratide containing the N48 deamidation hotspot after enzymolysis) using an Orbitrap high-resolution mass spectrometer to quantify deamidated cobratide. The limits of detection and quantification of this method reached 0.02 and 0.025 μM, respectively, and inter- and intra-day precision values of the method were <3%. The accuracy of the ¹⁸O-labelling strategy was validated by using samples containing synthesised peptides with a known ratio of deamidation impurities and also by comparing the final total deamidation results with our previously developed capillary electrophoresis method. The recoveries for deamidation (Asp), deamidation isomerisation (iso-Asp), and total deamidation were 101.52 ± 1.17, 102.42 ± 1.82, and 103.55 ± 1.07, respectively. The robustness of the method was confirmed by verifying the chromatographic parameters. Our results demonstrate the applicability of the ¹⁸O-labelling strategy for detecting protein deamidation and lay a robust foundation for protein therapeutics studies and drug quality consistency evaluations. Full article

(This article belongs to the Special Issue Analytical Applications of Biochemical Mass Spectrometry)

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10 pages, 1690 KiB

Open AccessArticle

Comparison of LC-MS³ and LC-MRM Method for Quantifying Voriconazole and Its Application in Therapeutic Drug Monitoring of Human Plasma

by Wenbo Ren, Lei Yin, Gaixia Zhang, Taiyu Zhai and Jing Huang

Molecules 2022, 27(17), 5609; https://doi.org/10.3390/molecules27175609 - 31 Aug 2022

Viewed by 1167

Abstract

The TDM of voriconazole which exhibits wide inter-individual variability is indispensable for treatment in clinic. In this study, a method that high-performance liquid chromatography tandem mass spectrometry cubed (HPLC-MS³) is first built and validated to quantify voriconazole in human plasma. The [...] Read more.

The TDM of voriconazole which exhibits wide inter-individual variability is indispensable for treatment in clinic. In this study, a method that high-performance liquid chromatography tandem mass spectrometry cubed (HPLC-MS³) is first built and validated to quantify voriconazole in human plasma. The system is composed of Shimadzu Exion LC^TM UPLC coupled with a Qtrap 5500 mass spectrometer. The separation of voriconazole is performed on a Poroshell 120 SB-C18 column at a flow rate of 0.8 mL/min remaining 7 min for each sample. The calibration curves are linear in the concentration range of 0.25–20 μg/mL. Intra-day and inter-day accuracies and precisions are within 8.0% at three concentrations, and the recoveries and matrix effect are all within accepted limits. In terms of stability, there is no significant degradation of voriconazole under various conditions. The HPLC-MS³ and HPLC-MRM (multiple reaction monitoring) methods are compared in 42 patients with Passing–Bablok regression and Bland–Altman plots, and the results show no significant difference between the two methods. However, HPLC-MS³ has a higher S/N (signal-to-noise ratio) and response than the MRM. Finally, the HPLC-MS³ assay is successfully applied to monitor the TDM (therapeutic drug monitoring) of voriconazole in human plasma, and this verifies that the dosing guidelines for voriconazole have been well implemented in the clinic and patients have received excellent treatment. Full article

(This article belongs to the Special Issue Analytical Applications of Biochemical Mass Spectrometry)

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15 pages, 2286 KiB

Open AccessArticle

Proteome-Based Serotyping of the Food-Borne Pathogens Salmonella Enterica by Label-Free Mass Spectrometry

by Xixi Wang, Chen Chen, Yang Yang, Lian Wang, Ming Li, Peng Zhang, Shi Deng and Shufang Liang

Molecules 2022, 27(14), 4334; https://doi.org/10.3390/molecules27144334 - 06 Jul 2022

Cited by 1 | Viewed by 1494

Abstract

Food-borne diseases caused by Salmonella enterica of 2500 serovars represent a serious public health problem worldwide. A quick identification for the pathogen serovars is critical for controlling food pollution and disease spreading. Here, we applied a mass spectrum-based proteomic profiling for identifying five [...] Read more.

Food-borne diseases caused by Salmonella enterica of 2500 serovars represent a serious public health problem worldwide. A quick identification for the pathogen serovars is critical for controlling food pollution and disease spreading. Here, we applied a mass spectrum-based proteomic profiling for identifying five epidemiologically important Salmonella enterica subsp. enterica serovars (Enteritidis, Typhimurium, London, Rissen and Derby) in China. By label-free analysis, the 53 most variable serovar-related peptides, which were almost all enzymes related to nucleoside phosphate and energy metabolism, were screened as potential peptide biomarkers, and based on which a C5.0 predicted model for Salmonella enterica serotyping with four predictor peptides was generated with the accuracy of 94.12%. In comparison to the classic gene patterns by PFGE analysis, the high-throughput proteomic fingerprints were also effective to determine the genotypic similarity among Salmonella enteric isolates according to each strain of proteome profiling, which is indicative of the potential breakout of food contamination. Generally, the proteomic dissection on Salmonella enteric serovars provides a novel insight and real-time monitoring of food-borne pathogens. Full article

(This article belongs to the Special Issue Analytical Applications of Biochemical Mass Spectrometry)

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18 pages, 2780 KiB

Open AccessArticle

Rapid Characterization of Bacterial Lipids with Ambient Ionization Mass Spectrometry for Species Differentiation

by Hung Su, Zong-Han Jiang, Shu-Fen Chiou, Jentaie Shiea, Deng-Chyang Wu, Sung-Pin Tseng, Shu-Huei Jain, Chung-Yu Chang and Po-Liang Lu

Molecules 2022, 27(9), 2772; https://doi.org/10.3390/molecules27092772 - 26 Apr 2022

Cited by 2 | Viewed by 1963

Abstract

Ambient ionization mass spectrometry (AIMS) is both labor and time saving and has been proven to be useful for the rapid delineation of trace organic and biological compounds with minimal sample pretreatment. Herein, an analytical platform of probe sampling combined with a thermal [...] Read more.

Ambient ionization mass spectrometry (AIMS) is both labor and time saving and has been proven to be useful for the rapid delineation of trace organic and biological compounds with minimal sample pretreatment. Herein, an analytical platform of probe sampling combined with a thermal desorption–electrospray ionization/mass spectrometry (TD-ESI/MS) and multivariate statistical analysis was developed to rapidly differentiate bacterial species based on the differences in their lipid profiles. For comparison, protein fingerprinting was also performed with matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) to distinguish these bacterial species. Ten bacterial species, including five Gram-negative and five Gram-positive bacteria, were cultured, and the lipids in the colonies were characterized with TD-ESI/MS. As sample pretreatment was unnecessary, the analysis of the lipids in a bacterial colony growing on a Petri dish was completed within 1 min. The TD-ESI/MS results were further performed by principal component analysis (PCA) and hierarchical cluster analysis (HCA) to assist the classification of the bacteria, and a low relative standard deviation (5.2%) of the total ion current was obtained from repeated analyses of the lipids in a single bacterial colony. The PCA and HCA results indicated that different bacterial species were successfully distinguished by the differences in their lipid profiles as validated by the differences in their protein profiles recorded from the MALDI-TOF analysis. In addition, real-time monitoring of the changes in the specific lipids of a colony with growth time was also achieved with probe sampling and TD-ESI/MS. The developed analytical platform is promising as a useful diagnostic tool by which to rapidly distinguish bacterial species in clinical practice. Full article

(This article belongs to the Special Issue Analytical Applications of Biochemical Mass Spectrometry)

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15 pages, 5219 KiB

Open AccessArticle

Spherical Sampler Probes Enhance the Robustness of Ambient Ionization Mass Spectrometry for Rapid Drugs Screening

by Mariya A. Shamraeva, Denis S. Bormotov, Ekaterina V. Shamarina, Konstantin V. Bocharov, Olga V. Peregudova, Stanislav I. Pekov, Eugene N. Nikolaev and Igor A. Popov

Molecules 2022, 27(3), 945; https://doi.org/10.3390/molecules27030945 - 30 Jan 2022

Cited by 10 | Viewed by 3131

Abstract

Ambient ionization mass spectrometry has become one of the most promising approaches for rapid and high-throughput screening of small molecules in complex biological matrices for emergency medicine, forensics, and food and agriculture applications. The simple procedures for sample collection and ionization without additional [...] Read more.

Ambient ionization mass spectrometry has become one of the most promising approaches for rapid and high-throughput screening of small molecules in complex biological matrices for emergency medicine, forensics, and food and agriculture applications. The simple procedures for sample collection and ionization without additional pretreatment are vital in these fields. Many efforts have been devoted to modifying various ambient ionization techniques to simplify the procedures and improve the robustness and sensitivity of the methods. Here, we demonstrate the implementation of rigid spherical sampler probes to improve the robustness of touch spray ionization mass spectrometry. The sphericity of the probes increases the stability of the cone-jet mode of electrospray, reduces the requirements for fine positioning of a sampler in the ion source, and decreases the possibility of corona discharge occurrence. The utilization of spherical sampler probes allows fast, non-invasive sampling, followed by rapid analysis for various drugs of different chemical classes in complex biological matrices, such as the whole blood or sebum collected from the skin surface. The linearity of the analytical signal response from drug concentration confirms the possibility of creating a simple semiquantitative method for small molecules monitoring using spherical sampler probes. Full article

(This article belongs to the Special Issue Analytical Applications of Biochemical Mass Spectrometry)

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Review

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16 pages, 1224 KiB

Open AccessReview

MALDI-TOF/MS Analysis of Non-Invasive Human Urine and Saliva Samples for the Identification of New Cancer Biomarkers

by Carlo Zambonin and Antonella Aresta

Molecules 2022, 27(6), 1925; https://doi.org/10.3390/molecules27061925 - 16 Mar 2022

Cited by 11 | Viewed by 3242

Abstract

Cancer represents a group of heterogeneous diseases that are a leading global cause of death. Even though mortality has decreased in the past thirty years for different reasons, most patients are still diagnosed at the advanced stage, with limited therapeutic choices and poor [...] Read more.

Cancer represents a group of heterogeneous diseases that are a leading global cause of death. Even though mortality has decreased in the past thirty years for different reasons, most patients are still diagnosed at the advanced stage, with limited therapeutic choices and poor outcomes. Moreover, the majority of cancers are detected using invasive painful methods, such as endoscopic biopsy, making the development of non-invasive or minimally invasive methods for the discovery and fast detection of specific biomarkers a crucial need. Among body fluids, a valuable non-invasive alternative to tissue biopsy, the most accessible and least invasive are undoubtedly urine and saliva. They are easily retrievable complex fluids containing a large variety of endogenous compounds that may provide information on the physiological condition of the body. The combined analysis of these fluids with matrix-assisted laser desorption ionization–time-of-flight mass spectrometry (MALDI-TOF/MS), a reliable and easy-to-use instrumentation that provides information with relatively simple sample pretreatments, could represent the ideal option to rapidly achieve fast early stage diagnosis of tumors and their real-time monitoring. On this basis, the present review summarizes the recently reported applications relevant to the MALDI analysis of human urine and saliva samples. Full article

(This article belongs to the Special Issue Analytical Applications of Biochemical Mass Spectrometry)

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