No abstract

Among all chemical sameness characterization tests of Therapeutic Peptides (TPs), one of the most significant and challenging aspects is to demonstrate comparable impurity profiles (both qualitative & quantitative) between a generic product and reference listed drug (RLD).

Chiral HPLC methods for natural and unnatural Nα-Fmoc/Boc amino acid derivatives used in therapeutic peptides.

Herein, we report the identification, isolation, and characterization of two unidentified, unspecified degradant impurities of Naloxone hydrochloride injection USP, 2 mg/2 ml solution. These impurities are predominantly observed during the sixth month accelerated stability condition at 40°C/75%RH and found to be increased to the levels of 0.10% and 0.17%, respectively, by using high-performance liquid chromatography (HPLC) with UV detection. Enrichment of these impurities was successfully achieved in acidic stress conditions and the formation of these impurities, as indicated by HPLC, were found to be like 2.14% and 4.64%, respectively. Further the impurities were detected, identified, isolated, and characterized by using various analytical techniques such as gradient reverse-phase HPLC, liquid chromatography-tandem mass spectrometry, reverse-phase preparative liquid chromatography, nuclear magnetic resonance (NMR) and elemental analysis. The unknown impurities structures were proposed as (4R,4aS,7aR,12bS)-3allyl-4a,9-dihydroxy-2,3,4,4a,5,6-hexahydro-1H-4,12-methanobenzofuro[3,2-e] isoquinolin-7(7aH)-one (degradation impurity-I) and (4bS, 5R, 8aS, 9R)-11-allyl-3,4,5,8a-tetrahydroxy-8,8a,9,10-tetrahydro-5H-9,4b-(epiminoethano) phenanthrene-6, 7-dione (degradation impurity-II). Structural elucidation of these impurities was performed by onedimensional and two-dimensional spectral data (1H NMR, 13C NMR, DEPT and 1H-1HgDQFCOSY, gHMBC, gHSQC, 1H-1HgROESY, MS, MS/MS, and elemental composition). The most plausible mechanism for the formation of impurities I and II were discussed in details.

In this study, we report the systematic approach for characterization of two major degradant impurities, which are not listed in any compendia and were formed during the stability studies of Dihydroergotamine mesylate injection (DHE). An ion-pair UPLC chromatographic method was developed to quantify the related substances present in the DHE injection drug product. The same was used to monitor the impurity profiling during its stability. The two unknown impurities were observed at RRT about 0.08 (Impurity-1) and RRT about 0.80 (Impurity-5) and found to be significantly increasing on stability. Forced degradation studies revealed the nature of the impurity and conditions required for enriching them. A Mass compatible HPLC method was developed to quantify only these two impurities using 25% ammonia and formic acid in water. Their mass numbers were identified using LC MS/MS with triple quadruple mass spectrometer coupled with a HPLC. These two impurities were then isolated from enriched products using preparative HPLC. These impurities were then characterized using Mass and NMR analysis along with Q-TOF elemental analysis.