RRML - Rapid LC-MS/MS assay for the evaluation of hydroxyl radical generation and oxidative stress induction in vivo in rats
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Concept, Design & Programming
Dr. Adrian Man
   
 
Nr. 18(4)/2010
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Rapid LC-MS/MS assay for the evaluation of hydroxyl radical generation and oxidative stress induction in vivo in rats

Daniela-Saveta Popa, Laurian Vlase, Daciana Chirilă, Felicia Loghin


Abstract:

A new high-throughput liquid chromatography coupled with mass spectrometry (LC-MS/MS) method for the quantification of 2,3-dihydroxybenzoic acid (2,3-DHBA) and 2,5- dihydroxybenzoic acid (2,5-DHBA) in rat plasma was developed and validated. The separation was performed on a Luna HILIC column under isocratic conditions using a 94:6 (v/v) mixture of acetonitrile and 50 mM ammonium acetate in water (pH 4,5) at 15ºC with a flow rate of 0.5 mL/min. The detection was performed in MRM mode using an ion trap mass spectrometer with electrospray negative ionization. The ion transition monitored was m/z 153 → m/z (109) for both analytes. The rat plasma samples were precipitated using acetonitrile and aliquots from supernatants obtained after centrifugation were directly injected into the chromatographic system. The method showed a good linearity (r > 0.998), precision and accuracy (< 9.1%) over the concentration ranges of 20 - 2420 ng/mL for both analytes. The recoveries were between 85.7% – 103.0%. The method is not expensive, it needs a minimal time for plasma sample preparation and has a run-time of 4 min for instrument analysis (retention times were of 1.5 min for 2,3-DHBA, and of 2.0 min for 2,5-DHBA, respectively). The developed and validated LC-MS/MS method is very simple and more time-saving compared to other similar published methods. It was used in a toxicological assay for the study of the in vivo hydroxyl radical generation in rat after exposure to some xenobiotics and oxidative stress generation.

Keywords: 2,3-DHBA,hydroxyl radical,LC-MS/MS,oxidative stress,rat plasma

 
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How to cite
Popa DS, Vlase L, Chirilă D, Loghin F. Rapid LC-MS/MS assay for the evaluation of hydroxyl radical generation and oxidative stress induction in vivo in rats. Rev Romana Med Lab. 2010;18(4):37-45