Development of a Stability-Indicating Purity Method for Ubrogepant through Stress Degradation Analysis, Extraction, and Characterization of Unidentified Degradation Products Using Flash Chromatography, NMR, IR, and LC-MS.

BACKGROUND: Ubrogepant is a prescription medication used to prevent migraine headaches. It is currently available in tablet form. OBJECTIVE: The goal of this work is to investigate the drug degradation profile of Ubrogepant, as well as to isolate and characterize undiscovered Ubrogepant degradation products by utilizing LC-MS, NMR, and IR spectroscopic analytical techniques. Furthermore, to develop a high resolution, sensitive, stability-indicating analytical approach for detecting and quantifying Ubrogepant degradation products in its pharmaceutical formulation. METHOD: To identify and quantify the degradation products of Ubrogepant in pharmaceutical products, a novel gradient reverse-phase HPLC (RP-HPLC) technique with a PDA detector was developed by utilizing a C18 stationary phase column. The eluent comprised a mixture of acetonitrile and water with 0.1% v/v ortho-phosphoric acid. To establish the intrinsic stability of Ubrogepant pharmaceutical product, it was stress tested under various degrading circumstances, including water, alkaline, acid hydrolysis, photolytic, oxidative, and thermal. Flash chromatography was used to isolate the two major degradants, and the structures were determined using NMR (1H , 13C, DEPT 135), IR, and LC-MS methods. RESULTS: The Ubrogepant medication was relatively more degradable in alkaline and acidic circumstances, and two unique degradation products were discovered. Based on spectroscopic and chromatographic evidence, it was conclusively demonstrated that these unique compounds were Ubrogepant hydrolysis products. All degradation products have been separated with a resolution greater than 2.0. The peak purity data shown that the Ubrogepant peak in all of the stress samples examined was pure. Under all stress environments, Ubrogepant achieved a minimum mass balance of 95%. The validated developed approach was sensitive enough to quantify Ubrogepant degradation products at 0.03% of the Ubrogepant test concentration. CONCLUSIONS: The proposed method was found to be stability-indicating since it fits all of the regulatory authorities' typical requirements. This method is highly efficient for detecting and quantifying impurities in Ubrogepant drug substances and drug products in QC laboratories. HIGHLIGHTS: Two new degradation products of Ubrogepant were successfully extracted and characterized using NMR, IR, and LC-MS spectroscopic methods. The proposed HPLC method can accurately quantify the degradation products of Ubrogepant in pharmaceutical products and is sensitive enough to detect degradation products of Ubrogepant as low as 0.17 µg/mL.

Evaluation of effects of temperature and humidity on the secondary structure of Ganirelix in an injectable formulation and comparison with Orgalutran® using circular dichroism spectroscopy.

Ganirelix, a drug used in in vitro fertilization (IVF), prevents ovulation in women who are not ready to have children by inhibiting a gene that produces gonadotropin. Peptides are macromolecules that are able to preserve a predetermined shape while carrying out the structural and regulatory roles for which they were originally intended. Peptide structures can be altered in the production and storage processes. Therapeutic peptides' biological activity can be drastically altered by even small modifications in their primary and secondary structures. The molecules' secondary structures can be monitored by subjecting them to different processing or storage conditions. In our investigation, we used circular dichroism (CD) spectroscopy with two different software programs for secondary structure evaluation to look at how environmental factors like temperature and humidity affected the secondary structure of Ganirelix in an injectable formulation. The CD results revealed that the alpha-helical (regular and distorted), beta-sheet, beta-strands (regular and distorted), beta-turn, and random coil structures of temperature and humidity stressed generic drug products are comparable to reference-listed drug.

Establishment of validated stability indicating purity method based on the stress degradation behavior of gonadotropin-releasing hormone antagonist (ganirelix) in an injectable formulation using HPLC and LC-MS-QTOF.

Stress study of a drug substance or pharmaceutical drug product provides a vision into degradation pathways and degradation products of the active pharmaceutical ingredient and helps in interpretation of the chemical structure of the degradation impurities. In the current study, Ganirelix active ingredient presented in the Orgalutran® was stressed with acidic and alkali hydrolysis, photolysis, thermal and oxidation conditions as per the guidelines of International Conference on Harmonization (ICH) Q1A (R2). Ganirelix was found to be labile under thermal and alkali hydrolytic stress conditions, while it was stable to acid hydrolytic, oxidative and photolytic stress. All degradation products were separated with a resolution > 1.5 on a C18 column (2.6 µm, 25 cm×4.6 mm) using a hydrophilic ion pair such as sodium perchlorate, at a concentration <0.04 M. In total, four major degradant impurities were found during stress study. These impurities were fractionated and desalted by flash chromatography for identification of chemical structures. LC-MS-QTOF analysis revealed that two degradation products are diastereomers of Ganirelix, one degradation product is a deamination compound and other degradation product result from the insertion of a new amino acid residue in the Ganirelix peptide sequence. The developed method is sensitive enough to quantify the related substances of Ganirelix at the 0.04% level with that of Ganirelix test concentration.

Evaluation of temperature, excipients impact on the primary structure of Ganirelix in an injectable formulation, and comparison with Orgalutran® using MALDI-TOF/TOF-MS.

Ganirelix is a linear polypeptide consisting of covalently bonded 10 amino acid residues. The amino acid sequence in a peptide determines the properties of the molecule. The slightest change in the primary structure (amino acid sequence) of therapeutic peptides can significantly impact its safety, efficacy, and immunogenicity. Hence, the primary structure analysis of therapeutic peptides is regarded as a critical quality attribute (CQA). A vast array of analytical techniques can be used to capture the primary structure of the peptide. In this study, we applied matrix-assisted laser desorption ionization (MALDI)/tandem time of flight mass spectroscopic (TOF/TOF MS) method to demonstrate the primary structure of Ganirelix in an injectable formulation. The apparent monoisotopic molecular mass of Ganirelix is 1,568.9 Da. The attained primary amino acid sequence of Ganirelix in temperature-stressed generic product matched with the theoretical sequence and showed homology with those of the reference listed drug (RLD).

Determination of Amino Acid Composition of Ganirelix Acetate in an Injectable Formulation by Pre-column Derivatization with 6-Aminoquinolyl-N-hydroxysuccinimidyl Carbamate.

Ganirelix is a synthetic decapeptide linked with nine different amino acids. To understand the peptide amino acid sequence or primary structure, the first step is to determine the amino acid composition of the peptide which can be a determining factor for the peptide immunogenicity. Edman degradation is not a suitable analytical technique to identify amino acid sequence present in Ganirelix due to the absence of uncharged N-terminal amino group. To address this challenge, a pre-column derivatization method was developed with 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate reagent. In the present work, the Ganirelix active pharmaceutical ingredient present in the injectable formulation was isolated by fraction collection and further purified by flash chromatography. The amino acid composition of Ganirelix is assayed by carrying out acid hydrolysis with 6 mol L-1 hydrochloric acid solution containing 1% phenol at 100°C for 24 h and derivatization with 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate reagent solution, followed by determination of individual amino acids by reverse-phase chromatography using a C18 column. High resolution was achieved for the nine amino acid mixture. The amino acid composition results of temperature-stressed Ganirelix generic product and reference listed drug are in good agreement with the theoretical molar ratio of label information.