Analysis of unstable degradation impurities of a benzodiazepine and their quantification without isolation using multiple linear regression.

This manuscript presents a novel methodology for calculating the relative response factors (RRFs) of unstable degradation impurities of molibresib (1). The degradation impurities were observed by HPLC during stress testing and were accompanied by large mass balance deficits. However, the impurities could not be isolated for traditional RRF determination due to their instability. The RRFs of two degradation impurities were determined without isolation by multiple linear regression analysis of HPLC-UV data. The results permitted accurate quantification of the degradants. The benefits and drawbacks of the approach are discussed, including suggested validation acceptance criteria.

The Degradation Chemistry of Farglitazar and Elucidation of the Oxidative Degradation Mechanisms.

The chemical degradation of farglitazar (1) was investigated using a series of controlled stress testing experiments. Farglitazar drug substance was stressed under acidic, natural pH, basic, and oxidative conditions in solution. In the solid state, the drug substance was stressed with heat, high humidity, and light. Farglitazar was found to be most labile toward oxidative stress. A series of mechanistic experiments are described in which the use of 18O-labelled oxygen demonstrated that oxidative degradation of farglitazar is caused primarily by singlet oxygen formed under thermal conditions. Major degradation products were isolated and fully characterized. Mechanisms for the formation of degradation products are proposed. Drug product tablets were also stressed in the solid state with heat, high humidity, and light. Stressed tablets afforded many of the same degradation products observed during drug substance stress testing, with oxidation again being the predominant degradation pathway. Evidence for the activity of singlet oxygen, formed during thermal stress testing of the solid oral dosage form, is presented. The degradation pathways observed during stress testing matched those observed during long-term stability trials of the drug product.

Simultaneous quantitation of trace level hydrazine and acetohydrazide in pharmaceuticals by benzaldehyde derivatization with sample 'matrix matching' followed by liquid chromatography-mass spectrometry.

Hydrazine and acetohydrazide are potential genotoxins and therefore need to be controlled in APIs and drug products to ppm levels for patient safety in cases where there is a reasonable probability of either of them being present. They are structurally related and could both be formed in the same chemical process under certain circumstances. However, no previous studies have reported simultaneous trace level quantification of these two compounds. Herein, a chemical derivatization scheme using benzaldehyde followed by LC-MS analysis is presented to address that need. During method development, unexpectedly high recoveries were encountered and presented a major challenge. A systematic investigation was undertaken to understand the benzaldehyde derivatization reaction and determine the underlying causes of the unacceptable recovery. It was found that this was due to the presence of the counter ion of the API in the sample matrix. Employing a 'matrix matching' sample preparation strategy, which involved acidifying the derivatization reaction medium with benzoic acid, gave similar reaction rates for the chemical derivatization in the presence and absence of the API salt and accordingly more consistent recoveries. Resultantly, a robust method for simultaneous quantification of hydrazine and acetohydrazide (1-100ppm) was successfully developed and validated.

The utility of sulfonate salts in drug development.

The issue of controlling genotoxic impurities in novel active pharmaceutical ingredients (APIs) is a significant challenge. Much of the current regulatory concern, has been focused on the formation and control of genotoxic sulfonate esters. This is linked with the withdrawal of Viracept (Nefinavir mesilate) from European markets in mid-2007, over concerns about elevated levels of ethyl methanesulfonate (EMS). This issue has resulted in calls from European regulators to assess risk mitigation strategies for all marketed products employing a sulfonic acid counter-ion to ensure that the sulfonate esters that could be potentially formed are controlled to threshold of toxicological concern (TTC)-based limits. This has even led to calls to avoid sulfonic acids as salt counter-ions. However, sulfonic acid salts possess a range of properties that are useful to both synthetic and formulation chemists. Whilst sulfonate salts are not a universal panacea to some of the problems of salt formation they do offer significant advantages as alternatives to other salt forming moieties under certain circumstances. This review thus sets out to define some of the advantages provided through utilization of sulfonic acids, explaining the importance of their retention as part of a thorough salt selection process.

Elucidating the pathways of degradation of denagliptin.

Stress testing or forced degradation studies of denagliptin (1) tosylate in solution and solid-state, its blends with excipients, and capsules were conducted in order to elucidate degradation pathways, aid formulation development, and generate data to support regulatory filings. In solution, denagliptin was stressed in acid, water, and base using organic cosolvents. In the solid-state, denagliptin was stressed under heat, humidity, and light. Blends of denagliptin with various excipients were stressed under heat and humidity in order to evaluate whether tablet was a viable dosage form. Capsules were stressed under heat, humidity, and light. It was found that denagliptin was stable in the solid-state, but degraded in solution, in blends with all excipients, and in capsules predominantly by cyclization to (3S,7S,8aS) amidine (2), which epimerized to (3S,7S,8aR) amidine (3). (3S,7S,8aR) amidine (3) subsequently hydrolyzed to the corresponding diketopiperazine (4). The purpose of this manuscript is to discuss the results of stress testing studies conducted during the development of denagliptin and the elucidation of its key degradation pathway.

Development and validation of an automated static headspace gas chromatography-mass spectrometry (SHS-GC-MS) method for monitoring the formation of ethyl methane sulfonate from ethanol and methane sulfonic acid.

An automated sample preparation and analysis procedure was developed to monitor the formation of ethyl methane sulfonate from reaction mixtures containing ethanol and methane sulfonic acid. The system is based on a liquid handling robot combined with a static headspace module. The formed ethyl methane sulfonate is analysed after derivatisation with pentafluorothiophenol using static headspace-gas chromatography-mass spectrometry (SHS-GC-MS). Using the automated reaction-derivatisation-headspace GC-MS system, the formation of ethyl methane sulfonate can be monitored in different reaction mixtures under different reaction conditions, including temperature, water content and pH. Excellent linearity, repeatability and robustness were obtained, allowing the system to be used in kinetic studies.