Development and Validation of Multiple Reaction Monitoring (MRM) Assays for Clinical Applications

2019 ◽  
pp. 205-223 ◽  
Author(s):  
Georgia Kontostathi ◽  
Manousos Makridakis ◽  
Vasiliki Bitsika ◽  
Nikolaos Tsolakos ◽  
Antonia Vlahou ◽  
...  
Keyword(s):  
Multiple Reaction Monitoring ◽  
Clinical Applications ◽  
Reaction Monitoring ◽  
Development And Validation

scholarly journals Development and Validation of an HPLC-MS/MS Method for Rapid Simultaneous Determination of Cefprozil Diastereomers in Human Plasma

2018 ◽  
Vol 2018 ◽  
pp. 1-9 ◽  
Author(s):  
Guodong He ◽  
Liping Mai ◽  
Xipei Wang
Keyword(s):  
Mass Spectrometry ◽  
Antimicrobial Activity ◽  
Multiple Reaction Monitoring ◽  
Internal Standard ◽  
Reaction Monitoring ◽  
Reverse Phase ◽  
Development And Validation ◽  
Positive Ion ◽  
Sensitive Method

Background. Both cis- and trans-cefprozil have antimicrobial activity, but their potencies are quite different. It is therefore necessary to develop a sensitive method to simultaneously determine both isomers for pharmacokinetic and bioequivalence studies. Methods. An LC-MS/MS method, using stable isotope-labeled cefprozil as the internal standard, was developed and validated. The analytes were extracted from plasma by protein precipitation and separated on a reverse-phase C18 column using a gradient program consisting of 0.5% formic acid and acetonitrile within 4 min. The mass spectrometry acquisition was performed with multiple reaction monitoring in positive ion mode using the respective [M+H]+ ions, m/z 391.2→114.0 for cefprozil and 395.0→114.5 for cefprozil-D4. Results. The calibration curves were linear over the ranges of 0.025–15 μg/mL for cis-cefprozil and 0.014–1.67 μg/mL for trans-cefprozil. The accuracies for the cis and trans isomers of cefprozil were 93.1% and 103.0%, respectively. The intra- and interassay precisions for the QC samples of the isomers were < 14.3%. The intra- and interassay precisions at the LLOQ were < 16.5%. Conclusions. The method was sensitive and reproducible and was applied in a pilot pharmacokinetic study of healthy volunteers.

Development and Validation of a LC–MS/MS Method

2013 ◽  
Vol 722 ◽  
pp. 255-259
Author(s):  
Meng Wang ◽  
Wen Jia Zhou ◽  
Quan Ying Zhang ◽  
Ming Huang
Keyword(s):  
Mobile Phase ◽  
Ammonium Acetate ◽  
Multiple Reaction Monitoring ◽  
Tandem Mass ◽  
Reaction Monitoring ◽  
Limit Of Quantification ◽  
Chromatography Tandem Mass Spectrometry ◽  
Liquid Chromatography Tandem Mass ◽  
Positive Mode ◽  
Development And Validation

A simple, sensitive, selective, rapid, reproducible and specific liquid chromatography-tandem mass spectrometry (LC-MS/MS) method has been developed and validated for the identification and quantification of paroxetine (PAX) in human plasma,. Chromatographic separation was performed on XTerra RP18 (5 μm, 150 mm × 4.6 mm i.d.) column with mobile phase composed of 10 mM ammonium acetate containing 0.2% formic acid: methanol (30:70, v/v) at flow rate of 0.9 mL min-1. PAX and CZP were detected with proton adducts at m/z (amu) 330.1 192.1 and 327.2 270.1, in multiple reaction monitoring (MRM) positive mode. The method was validated over the concentration range of 0.05 - 30 ng mL-1. The lower limit of quantification (LLOQ) was 0.05 ng mL-1. The inter-run and intra-run precision was within 2.1-11.8% and 2.2-5.8%, respectively

scholarly journals PSV-25 Detection of heart and aorta tissue peptide markers by the multiple-reaction monitoring method

2020 ◽  
Vol 98 (Supplement_4) ◽  
pp. 362-363
Author(s):  
Daniil Khvostov ◽  
Natalya Vostrikova ◽  
Irina M Chernukha
Keyword(s):  
Amino Acid ◽  
High Performance ◽  
Multiple Reaction Monitoring ◽  
Meat Product ◽  
Meat Products ◽  
Amino Acid Sequences ◽  
Composition Analysis ◽  
Reaction Monitoring ◽  
Russian Science ◽  
Monitoring Method

Abstract Functional, particularly personalized meat-based foods are of more in demand by a consumer today. Functional additives, such as plant components and animal proteins from bovine or porcine tissues have been successfully used. With many ingredients added to foods, it is important to provide quality and composition monitoring to confirm the products’ authenticity, to identify undeclared or rarely used types of raw meat in product formulations. For example, if animal heart tissue is a component of a product formulation or if aorta tissue presents in a product due to improper trimming. Different methods are used to identify raw materials, including new approaches in proteomics and peptidomics that are considered the most effective modern methods nowadays. The purpose of the study is meat product composition analysis and special biomarker peptide identification to confirm the presence of heart and aorta tissue in a finished meat product. Over 20 amino acid sequences were checked based on earlier obtained data. Those amino acid sequences were analyzed with a high-performance liquid chromatography with mass spectrometric detection as described. The MS settings were selected using the Skyline. Signal-to-Noise ratio (S/N) over 10 units were used to choose the best peptide candidates. Seven peptides were found in porcine hearts. The best candidate was peptide VNVDEVGGEALGR (S/N - 73.10±5.3) from β-Hemoglobin. Two marker peptides from serum albumin were selected for pork aorta: TVLGNFAAFVQK (S/N 53.51±2.4) and EVTEFAK (S/N 31.69±4.1). These biomarkers showed the best detection and specificity. The multiply reaction monitoring method made it possible to identify the most/best specific peptides—biomarkers that could confirm the heart and/or aorta in meat products. The method can be used for comparative research or identification of best peptides that are specific to any type of animal tissue. The work was supported by the Russian Science Foundation, project no. 16-16- 10073.

scholarly journals Calcineurin Activity Assay Measurement by Liquid Chromatography–Tandem Mass Spectrometry in the Multiple Reaction Monitoring Mode

2014 ◽  
Vol 60 (2) ◽  
pp. 353-360 ◽  
Author(s):  
Lynn Carr ◽  
Anne-Laure Gagez ◽  
Marie Essig ◽  
François-Ludovic Sauvage ◽  
Pierre Marquet ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Liquid Chromatography ◽  
Mononuclear Cells ◽  
Multiple Reaction Monitoring ◽  
Internal Standard ◽  
Reaction Monitoring ◽  
Activity Assay ◽  
Peripheral Blood Mononuclear ◽  
Blood Concentrations ◽  
Transplant Patients

Abstract BACKGROUND Blood concentrations of the calcineurin inhibitors (CNIs) cyclosporine and tacrolimus are currently measured to monitor immunosuppression in transplant patients. The measurement of calcineurin (CN) phosphatase activity has been proposed as a complementary pharmacodynamic approach. However, determining CN activity with current methods is not practical. We developed a new method amenable to routine use. METHODS Using liquid chromatography–multiple reaction monitoring mass spectrometry (LC-MRM-MS), we quantified CN activity by measuring the dephosphorylation of a synthetic phosphopeptide substrate. A stable isotope analog of the product peptide served as internal standard, and a novel inhibitor cocktail minimized dephosphorylation by other major serine/threonine phosphatases. The assay was used to determine CN activity in peripheral blood mononuclear cells (PBMCs) isolated from 20 CNI-treated kidney transplant patients and 9 healthy volunteers. RESULTS Linearity was observed from 0.16 to 2.5 μmol/L of product peptide, with accuracy in the 15% tolerance range. Intraassay and interassay recoveries were 100.6 (9.6) and 100 (7.5), respectively. Michaelis–Menten kinetics for purified CN were Km = 10.7 (1.6) μmol/L, Vmax = 2.8 (0.3) μmol/min · mg, and for Jurkat lysate, Km = 182.2 (118.0) μmol/L, Vmax = 0.013 (0.006) μmol/min · mg. PBMC CN activity was successfully measured in a single tube with an inhibitor cocktail. CONCLUSIONS Because LC-MRM-MS is commonly used in routine clinical dosage of drugs, this CN activity assay could be applied, with parallel blood drug concentration monitoring, to a large panel of patients to reevaluate the validity of PBMC CN activity monitoring.

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