Original Articles: 2017 Vol: 9 Issue: 7
A Validated RP-HPLC Method for the Determination of 2-Chloroadenosine As Process Related Impurity in Regadenosen Parenteral Dosage Form
Regadenoson is an A2A adenosine receptor agonist that is a coronary vasodilator. Regadenoson is chemically described as adenosine, 2-[4[(methylamino) carbonyl]-1H-pyrazol-1-yl]-, monohydrate. Regadenoson is a low affinity agonist (Ki ≈ 1.3 μM) for the A2A adenosine receptor, with at least 10-fold lower affinity for the A1 adenosine receptor (Ki > 16.5 μM), and weak, if any, affinity for the A2B and A3adenosine receptors. Activation of the A2A adenosine receptor by regadenoson produces coronary vasodilation and increases coronary blood flow (CBF). A simple and precise reverse phase high performance liquid chromatography (RP-HPLC) method has been developed and validated which can separate and accurately quantitate 2-chloro adenosine related compounds. The capability of the method was demonstrated through adequate separation of all potential 2-chloro adenosine related compounds from active pharmaceutical ingredient and also from each other that are present in aged and stress degraded 2-chloro adenosine stability samples. Successful separation of 2-chloro adenosine from its one process impurity and degradation impurities formed under stress conditions was achieved by using a isocratic elution at a flow rate of 1.0 mL/min on a symmetry C18-3v column (250 mm × 4.6 mm, 5 μm particle size, 100 Å pore size) at ambient temperature. Mobile phase is the mixture of 1:1 v/v of acetonitrile and methanol; (pH-adjusted to 3.2 with 10% v/v ortho-phosphoric acid) in gradient mode was found to be best suitable for separation of impurity. UV detection at 205 nm was employed to monitor the analytes. The method was successfully validated in accordance to ICH guidelines acceptance criteria for system suitability, specificity, linearity, range, precision, accuracy, limits of detection and quantification for the impurities, and robustness, following the ICH guidelines. Therefore, the proposed method was suitable for the simultaneous determination of 2-chloro adenosine and its process related impurities. Finally, the applicability of the method was evaluated in commercial dosage form analysis as well as in stability studies.