Simultaneous Determination of Kynurenine, Fumaric acid and Pyroglutamic acid as Degradation Products in Amino acids Formulation

Abstract

The aim of the work was to develop and validate a HPLC method to quantify the Kynurenine (Kyn), Fumaric acid (Fum) and Pyroglutamic acid (Pyr) in amino acids (AAs) formulation as degradation products. Kyn, Fum and Pyr are the degradation products of Tryptophan (Trp), Aspartic acid (Asp) and Glutamic acid (Glu) respectively. Protein plays a crucial role in almost all biological processes and AAs are the building blocks of proteins. Trp is an essential amino acid, whereas Asp and Glu are non-essential AAs. Separation was main challenge due to non-significant absorption of UV radiation and zwitterion nature of AAs. However, separation was achieved using YMC C18AQ (4.6×250mm) 5µm column at 45°C with Waters Alliance e2695 HPLC equipped with PDA detector at 210 nm with gradient mobile phase consisting of phosphate buffer (pH 2.1) and acetonitrile as mobile phase A (98:2) and Acetonitrile as mobile phase B at a flow rate of 0.8 mL/min. This method shows excellent linear response with correlation coefficient (R > 0.9995) for all the three degradation products. The percentage recovery of three degradation products (Kyn, Fum and Pyr) in Amino acid formulation were found to be within the acceptance limit of 97 to 103 %. The precision of the method was less than the maximum allowable limit of percentage of relative standard deviation (% RSD) was = 2. Forced degradation of the drug product was carried out as per ICH guidelines with a view of stability indicating property of the method. The validated dosing range covered from 0.2 mg/L to 4 mg/L of Kyn; 0.1 mg/L to 1.5 mg/L of Fum; and 1.0 mg/L to 80 mg/L of Pyr. The developed method could be presented as a suitable method for separation and quantification of these degradation products in AAs formulations. The method specificity, accuracy, precision, linearity and robustness were proved in validation. The developed and validated HPLC method is simple, realistic and suitable for monitoring of Kyn, Fum, and Pyr in AAs formulations.