scholarly journals An Intact ACTH LC-MS/MS Assay as an Arbiter of Clinically Discordant Immunoassay Results

2019 ◽  
Vol 65 (11) ◽  
pp. 1397-1404 ◽  
Author(s):  
Junyan Shi ◽  
Pawan Dhaliwal ◽  
Yu Zi Zheng ◽  
Terry Wong ◽  
Joely A Straseski ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Adrenocorticotropic Hormone ◽  
Epitope Mapping ◽  
Multiple Reaction Monitoring ◽  
Method Comparison ◽  
Biologically Active ◽  
Chromatography Tandem Mass Spectrometry ◽  
Ms Analysis ◽  
Liquid Chromatography Tandem Mass ◽  
Active Fragments

Abstract BACKGROUND Measurement of plasma adrenocorticotropic hormone (ACTH) is key in the differential diagnosis of hypothalamic-pituitary-adrenal disorders. Two-site sandwich immunoassays dominate clinical testing of ACTH in North America; however, discordant results between manufacturers have been repeatedly reported. To resolve the discrepancy, we developed a liquid chromatography–tandem mass spectrometry (LC-MS/MS) assay for the intended measurand, biologically active intact ACTH (iACTH). METHODS The multiple reaction monitoring LC-MS/MS assay was designed to selectively measure full-length iACTH, as well as ACTH analogs and fragments (i.e., ACTH1–24 and ACTH18–39). Epitope assignment of the Roche Elecsys antibodies was performed by MALDI-TOF mass spectrometry. A method comparison between Roche Elecsys and Siemens Immulite ACTH immunoassays was performed and clinically concordant/discordant results identified. In a subset of these samples, the iACTH concentration was determined using the LC-MS/MS method. RESULTS The lower limit of the measuring interval of the iACTH LC-MS/MS assay was 9 pg/mL (2 pmol/L). The assay was linear from 9 to 1938 pg/mL (2 to 427 pmol/L). Epitope mapping revealed that the Roche capture and detection antibodies bound residues 9–12 and 36–39 of ACTH, respectively. The iACTH LC-MS/MS analysis demonstrated that for discordant results between 2 immunoassays studied, only the Roche results were highly positively correlated with the iACTH concentration. CONCLUSIONS Immunoprecipitation of biologically active ACTH molecules followed by LC-MS/MS analysis enabled selective detection of iACTH and relevant biologically active fragments in plasma. Applied to the investigation of clinically discrepant results, this method can act as an arbiter of the concentration of iACTH present.

scholarly journals A Hydrophilic Interaction Liquid Chromatography–Tandem Mass Spectrometry Quantitative Method for Determination of Baricitinib in Plasma, and Its Application in a Pharmacokinetic Study in Rats

Molecules ◽  
2020 ◽  
Vol 25 (7) ◽  
pp. 1600 ◽  
Author(s):  
Essam Ezzeldin ◽  
Muzaffar Iqbal ◽  
Yousif A. Asiri ◽  
Azza A Ali ◽  
Prawez Alam ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Liquid Chromatography ◽  
Tandem Mass Spectrometry ◽  
Multiple Reaction Monitoring ◽  
Internal Standard ◽  
Janus Kinase ◽  
Pharmacokinetic Studies ◽  
Tandem Mass ◽  
Chromatography Tandem Mass Spectrometry ◽  
Liquid Chromatography Tandem Mass

Baricitinib, is a selective and reversible Janus kinase inhibitor, is commonly used to treat adult patients with moderately to severely active rheumatoid arthritis (RA). A fast, reproducible and sensitive method of liquid chromatography-tandem mass spectrometry (LC-MS/MS) for the quantification of baricitinib in rat plasma has been developed. Irbersartan was used as the internal standard (IS). Baracitinib and IS were extracted from plasma by liquid–liquid extraction using a mixture of n-hexane and dichloromethane (1:1) as extracting agent. Chromatographic separation was performed using Acquity UPLC HILIC BEH 1.7 µm 2.1 × 50 mm column with the mobile phase consisting of 0.1% formic acid in acetonitrile and 20 mM ammonium acetate (pH 3) (97:3). The electrospray ionization in the positive-mode was used for sample ionization in the multiple reaction monitoring mode. Baricitinib and the IS were quantified using precursor-to-production transitions of m/z 372.15 > 251.24 and 429.69 > 207.35 for baricitinib and IS, respectively. The method was validated according to the recent FDA and EMA guidelines for bioanalytical method validation. The lower limit of quantification was 0.2 ng/mL, whereas the intra-day and inter-day accuracies of quality control (QCs) samples were ranged between 85.31% to 89.97% and 87.50% to 88.33%, respectively. Linearity, recovery, precision, and stability parameters were found to be within the acceptable range. The method was applied successfully applied in pilot pharmacokinetic studies.

scholarly journals PATH-19. TUMOR HETEROGENEITY IN GLIOMAS: A PILOT STUDY OF HISTOPATHOLOGY-ASSOCIATED PROTEOME PROFILES ASSESSED BY LIQUID CHROMATOGRAPHY TANDEM MASS SPECTROMETRY OF FFPE SAMPLES

2019 ◽  
Vol 21 (Supplement_6) ◽  
pp. vi147-vi147
Author(s):  
Aline Paixao Becker ◽  
Erica Hlavin Bell ◽  
S Jaharul Haque ◽  
Joseph McElroy ◽  
Jessica Fleming ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Liquid Chromatography ◽  
Tandem Mass Spectrometry ◽  
Tumor Heterogeneity ◽  
High Expression ◽  
Tandem Mass ◽  
Chromatography Tandem Mass Spectrometry ◽  
Ms Analysis ◽  
Ffpe Samples ◽  
Liquid Chromatography Tandem Mass

Abstract Herein, we aimed to scrutinize tumor heterogeneity of infiltrative gliomas based on histopathological phenotypes, through proteomic profiling of formalin-fixed, paraffin embedded (FFPE) tissue. FFPE tissues are promising samples for proteomic studies, which can support the elucidation of glioma evolution and identify therapeutically vulnerable proteins and signaling pathways that drive recurrence and resistance mechanisms. We represented 2–3 adjacent, phenotypically distinct areas from 12 grade II-IV gliomas diagnosed according to the 2016 WHO classification, in a total of 35 samples (1.0mm cores), that were analyzed employing liquid chromatography tandem mass spectrometry (LC-MS/MS) for label-free expression proteomics. The statistical analysis was performed using R and Qlucore™ omics explorer software. Overall, 9222 peptides were mapped to 1758 non-redundant proteins, 320 of which had a significant (p< 0.05) differential expression in glioblastomas versus lower grade gliomas (Wilcoxon test comparing average expression). Principal component analysis (PCA) of the whole set of proteins showed clustering of the samples by tumor grade and IDH status. Unsupervised hierarchical analysis of the most significantly expressed proteins (p= 0.01, FDR= 0.05) showed that IDHwt gliomas had high expression of proteins related to cell movement, DNA structure, and fatty acid metabolism throughout the samples. IDHmut gliomas largely displayed high expression of mitochondrial enzymes related to energy production and neurotransmitter metabolism, with subsets closely related to 1p19q status and histological grade. Importantly, we demonstrated that LC-MS/MS analysis of FFPE core samples is feasible and enables recognition of different proteome signatures across histopathological phenotypes within a single tumor. This is the first study, to our knowledge, exploring proteome profiles addressing histopathological heterogeneity in gliomas by LC-MS/MS analysis of FFPE samples, which warrants further validation in independent datasets including ones that utilize frozen specimens. FUNDING: R01CA108633, R01CA169368, RC2CA148190, U10CA180850-01 (NCI), Brain Tumor Funders Collaborative Grant, and the Ohio State University CCC (all to AC).

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