An improved and sensitive method for nitrotyrosine estimation by reverse phase-high performance liquid chromatography

Reactive nitrogen species such as peroxynitrite anion (ONOO-), nitrogen oxide radical (NO.) may lead to nitrotyrosine formation by oxidative nitration of tyrosine. Nitrotyrosine is considered as an important marker to estimate the severity of biological oxidative damage. Therefore, in the present study, we evaluated a reverse phase-high performance liquid chromatography (RP-HPLC) method to improve the sensitivity and specificity of the nitrotyrosine estimation. The mobile phase used in the present study was methanol (pH 4.7) and 0.1% phosphoric acid (60:40%). The standard serial dilutions were injected onto the HPLC column (C-18) at a flow rate of 1 ml/min mobile phase with a time window of 6 min. The results were monitored on the Diode Array detector at wavelength 210 nm. The evaluation and quantification of output signals were made on the Chromelion software version 6.80 which controls the whole liquid chromatography system. The same standard serial dilutions were monitored spectrophotometrically at 210nm. We found the standard curves were linear over the concentration range of 0.3 to 20 ng/ml for both RP-HPLC with UV detector (r2 = 0.997) and spectrophotometric (r2 = 0.999) methods.  In spectrophotometric method, only four standard points were detected out of seven and represented on a straight line, this shows the spectrophotometric method was unable to measure less than 2.5 ng/ml concentration. On the other hand, in RP-HPLC method, all the seven standards were lying on a straight line and HPLC machine can detect up to 1ng/ml or 1pg/µl. The present study established the highly specific, sensitive, accurate and cost-effective RP-HPLC with UV detector method for nitrotyrosine estimation.