Analytical Method Development for a Chiral Triazole Analogue
Challenge: To develop and validate a chiral HPLC method capable of monitoring the purity of a Triazole analogue in a novel production process and two other methods: LC/MS/MS for identification of process impurities; and a head space GC method capable of qualitatively and quantitatively monitoring residual organic volatile impurities (OVI) during the manufacturing process.
Technological procedures to scale up the production of the novel antifungal agent have recently been optimized. The scale up process has resulted in a change in the impurity profile of the molecule and therefore previously developed and validated HPLC methods were found to be inadequate for monitoring purity of the drug substance.
Solution: The disadvantage of chiral synthesis is that the separation cannot be easily scaled up. The first challenge was to select an appropriate chiral separation method for a new process. Three different methods of separating enantiomers by chromatography were evaluated:
- Form a diastereoisomeric derivative
- Use a chiral stationary phase
- Use a chiral mobile phase additive
Methods 1 and 2 have disadvantages in terms of the selectivity and purity of the derivatization agent and additives. The chromatography was difficult and took a considerable amount of development time. However, the main advantage was that the standard chiral stationary phases (column) could be used. The chiral stationary phases offered several advantages such as analytes would not need to be modified and the separations were rapid and efficient. The main disadvantage of these methods was they were costly to run. The other challenge was the selection of the most optimal chromatographic conditions. A number of factors that have a direct impact on the chiral separations were investigated.
Appropriate column selection
Chiral HPLC columns are made by immobilizing single enantiomers onto the stationary phase. Resolution relies on the formation of transient diastereoisomers on the surface of the column packing. The compound that forms the most stable diastereoisomer will be most retained, whereas the opposite enantiomer will form a less stable diastereoisomer and will elute first. To achieve discrimination between enantiomers there needs to be a minimum of three points of interaction to achieve chiral recognition.
The intermolecular forces involved with chiral recognition are polar/ionic interactions, pi-pi interactions, hydrophobic effects and hydrogen bonding. These can be increased by the formation of inclusion complexes and binding to specific sites such as receptor sites in complex phases. These intermolecular forces were manipulated by choosing suitable mobile phases including polar interactions which can be controlled by pH.
The effect of temperature is important in chiral HPLC methods. A lower temperature increases the chiral recognition, but as it alters the kinetics of mass transfer it actually makes the chromatography worse by broadening peaks. An optimum temperature was selected for a separation that allowed resolution of all chromatographic peaks.
During the course of this analytical research, a new HPLC method to determine the enantiomer and stereoisomer impurities of the triazole compound and several chiral degradation products was developed and validated. An appropriate column was selected for the chiral separation under polar ionic mode. To optimize resolution, several mobile phases containing different organic modifiers and salts were examined and the effect of the acid to base ratio on the resolution was also evaluated. The optimum conditions were obtained with a mobile phase containing acetonitrile / methanol / isopropanol / acetic acid / triethylamine solution. The HPLC separation was carried out on a ChiralPak AD-H, 5 micron, 4.6 x 250 mm and guard column Hypersil BDS (C18). The triazole-related chiral compounds were eluted using a Mobile Phase: 65:15:20 (Ethanol: Isopropyl alcohol: Ethyl Acetate).
The drug substance and its enantiomer, diastereoisomer impurities and degradation products were all separated. The method validation results revealed that the method was linear with a regression coefficient greater than 0.999. The recovery for these impurities was greater than 87%. The method specificity was also verified by injecting stressed drug substance solutions. No interference from potential degradation products was observed. The repeatability and intermediate precision was evaluated on the drug substance containing impurities at 0.1 to 0.2% levels. RSD's of less than 6% were achieved with each impurity measured. The method was found to be robust in terms of small changes in mobile phase composition and other HPLC parameters. The LOQ for the major degradation product was found to be 0.08%.
Following the completion of the method development work, a forced degradation study was conducted to demonstrate the method is capable of capturing all possible degradation products that might have appeared during scale up production. The forced degradation process had revealed that the method was not stability-indicating, i.e. could not detect all possible impurities or degradation products. As a result, further optimization of the newly developed method was performed through the selection of a suitable chromatographic column and again modified chromatographic conditions such temperature, mobile phase and gradient composition.
Results: This newly validated method was implemented successfully by the sponsor to monitor each impurity and degradation product in new drug product lots under different storage conditions to demonstrate product stability. No racemization of the molecule was observed by this method during the stability studies. Specificity was demonstrated by a spectrally pure analyte peak in the presence of degradation products from exposure to air, moisture, light, heat, acid, base, and oxidizer. The validated chiral assay is linear, precise, rugged, accurate and specific. It has been used to quantitatively assay lots from the scaled-up manufacturing process. The method is being now utilized for the in process control of the API manufacturing process as well as for the release of API.