Identification of Maillard Based Lactose Adduct in Roflumilast Generated During Incompatibility Studies and its RP-HPLC Method Development, Validation and Impurity Control

Abstract

Roflumilast is Prescribe as the first line treatment for COPD (Chronic Obstructive Pulmonary Disease). One of the critical starting materials for the synthesis of Roflumilast API is 3,5-dichloro-4-aminopyridine. It is also generated during the degradation of Roflumilast API and termed as degradant product-1(DP-1). The primary amino group of DP-1 reacts with lactose monohydrate (Maillard reaction) in the formulation to form DP-1Lactose Adduct (DPLA) under relatively harsh conditions. This adduct formed, was isolated and characterized by different spectroscopic methods. In-silico toxicological evaluation of characterized adduct was done using online free access software T.E.S.T toxicity estimation tool, Pro-TOX-II and Pre-ADMET, results advocating that the adduct may have Carcinogenicity, Developmental toxicity or Immuno-toxicity. In addition to this, a selective, robust and precise reversed-phase high-performance liquid chromatography (RP-HPLC) method was developed and validated as per ICH Q2(R1) guideline for the determination of Roflumilast, 3, 5-dichloro-4-aminopyridine in presence of DPLA. The chromatographic separation was performed with Phenomenex Luna C 18 column (250 mm × 4.6 mm, 5 μm) using a mobile phase consisting of Acetonitrile - Ammonium formate (10mM) (pH 8.0). pH was adjusted to 8 with triethylamine, in gradient mode 0 min (75:25, v/v), 3.5min (70:30, v/v) and 5 min (85:15, v/v) keeping the flow rate of 0.6 ml/min. Quantification was achieved using UV detection at 254nm. Linearity, accuracy, and precision were found to be under acceptable criteria for the determination of Roflumilast, 3, 5-dichloro-4-aminopyridine and DPLA in the concentration range of 200-1200 µg ml -1 , 1–6 µg ml -1 and 0.2-1.2 µg ml -1 , respectively. Further for rationalization of formation of DPLA, stability studies of DP-1 spiked in-house and marketed formulation were performed at 25°C ± 2°C and 60% RH ± 5% RH and at 40°C ± 2°C and 75% RH ± 5% RH, which conclude that up to the 180 days there was no evidence of the formation of lactose adduct so far. The DPLA should be controlled ≤ 0.3% during formulation development, calculated concerning the ICH M7 guideline to prevent its genotoxic effect. Overall, this study suggests either replacing the lactose monohydrate as an excipient in tablet or avoidance of high moisture and higher temperature during formulation development to avoid the impurity generation.