Improving the Quantitation of Unknown Impurity Analysis Using Dual-Gradient HPLC with Charged Aerosol Detection

Quantification of substances such as drug impurities or library compounds when pure standards are unavailable is difficult yet often necessary. This is often accomplished by HPLC based on relative response using low wavelength UV detection. The dependence of response on the optical properties of each component can lead to large errors in estimated quantity. Charged aerosol detection is a mass sensitive detection technique with near uniform response for all nonvolatile analytes, provided that the eluent remains constant. However, response changes during gradient conditions are common with all nebulization-based detectors. The use of inverse gradient postcolumn addition can effectively normalize responses. Using a single platform capable of dual gradient HPLC or UHPLC combined with charged aerosol detection allowed a group of compounds ranging in chemical structure and properties, UV absorbance, HPLC retention, and application in the pharmaceutical industry to be studied. The response deviation was significantly decreased across the compound set to ~13% compared to the 46% without the inverse gradient applied, and >60% with UV-based detection. The work demonstrated very good correlation in the linear response curves over the range tested. This allows for a single calibrant to be used to calculate the mass concentration of unknown impurities independent of their optical properties. This fully integrated system can be used to improve accuracy for mass balance calculations, analysis of impurities and degradants, monitoring compound synthesis and quality of library compounds, and cleaning validations while providing significant cost and time savings with identification and individual standard approaches. INTRODUCTION Interest in metabolite or trace impurity analysis in pharmaceutical industries is intensifying due to concerns with mass balance studies, regulatory commitments in reporting API impurities, metabolite in safety testing (MIST), and cleaning validation of manufacturing equipment. Most often an analytical requirement for accurately reporting the level of metabolites or impurities is to obtain reference standards. Since many of these standards remain unavailable it makes exact quantification of impurities and metabolites difficult. The situation is further compounded since several types of HPLC detectors such as UV or evaporative light scattering detection (ELSD) either lack the sensitivity to detect these compounds or do not provide uniform response across the target analytes. The development of cleaning validation methods is an area facing similar challenges. The need for a fast turn-around-time of the cleaned equipment to help maintain production schedules does not allow for identification of every peak present. So quantitation of impurities by UV detection is often done on a peak area bases. The difficulty that can be encountered when using a specific technique like HPLC-UV is how to quantify unknown peaks. UV detectors suffer from varying extinction coefficients for different structures and thus peak area percent calculations can result in significant errors in impurity calculations. Considering the major difference in UV response between an aromatic active ingredient and a non-aromatic surfactant such as dodecylsulfate, this can result in a potential source of significant underestimation of surfactant contamination. Another HPLC detection technique, ELSD often lacks sufficient sensitivity for trace analysis and due to the need to optimize methods for different compounds considerable response factor variation can occur even for compounds within a similar class structure. The Corona® Charged Aerosol Detector (CAD®) is mass sensitive and can be added to the traditional HPLC-UV platform. This detector provides the most consistent response across all nonvolatile and some semivolatile analytes of all HPLC detection techniques.1 When running gradients from high aqueous to high organic content all nebulizer-based detectors tend to show increased response as the organic solvent proportion increases due to improved nebulization efficiency. Aerosol-based detection techniques using CAD are also sensitive to this phenomen. Optimization of the detector response by delivering a second postcolumn solvent stream, which is inverted in composition relative to the elution gradient, enables a constant proportion of organic solvent to reach the detector and results in more uniform response factors for all compounds eluting from the column.2,3,4