The objective of this work was to develop a universal high performance liquid chromatography method that is capable of simultaneously retaining and separating both cations and anions within a single chromatographic analysis for the purpose of quantification in pharmaceutical products. A zwitterionic stationary phase operated in the hydrophilic interaction chromatography (HILIC) mode in conjunction with evaporative light scattering detection was investigated for the separation and quantitation of 33 commonly used pharmaceutical counter ions, 12 cations, and 21 anions. Using a single gradient chromatographic analysis, both anions and cations were easily separated from each other in addition the parent pharmaceutical molecules also were separated. The zwitterionic stationary phase utilized in this study offers unique separation capabilities based upon its mixed-mode separation mechanism (that is, electrostatic ion chromatography with the positively and negatively charged functional groups on the stationary phase and HILIC). As a result, a generic screening method was devised that allows for counterion determinations regardless of the pharmaceutical salt that is investigated. The unique retention characteristics of this column were evaluated by varying key mobile phase parameters, such as pH, buffer strength, and organic modifier. After examining the changes in retention, response, and resolution, this universal method was then further evaluated for reproducibility for multiple counterion determinations. For counterion determinations, a typical precision of <2.0% was observed for all counterions and most determinations were within 2.5% of the theoretical salt concentration. Thus, a very rugged screening method was developed capable of separating both anions and cations within a single chromatographic analysis. Counterion determinations were demonstrated for 10 pharmaceutically relevant salts.

The separation and quantitation of counterions in the pharmaceutical industry is an important determination. During drug development, the selection of the correct salt form early in the development process can prevent repeating toxicology, biological, and stability studies. As a result, development timeline delays can potentially be prevented. The initiation of the salt selection process generally takes place for all ionizable compounds that successfully have passed initial toxicology screening. The most common pharmaceutical salt forms are sodium salts of acids and hydrochloride salts of amines. Ideally, these salts would be nonhygroscopic, exhibit solid–state stability, and possess high aqueous solubility. However, the most common salt forms do not always possess the best physicochemical properties and attributes for development success. In these cases, a multidisciplinary salt-selection process is necessary to find alternative acceptable salt forms. Automated salt selection systems can be used to screen numerous counterions in various solvent systems, which can result in atypical salt forms. The salt forms that are crystalline from this screen will be scaled up for further evaluation. At this point, the analyst typically evaluates the salt forms using high performance liquid chromatography (HPLC) for counterion identity and stoichiometry confirmation. The final salt that proceeds into clinical trials typically has desirable properties in relation to stability, bioavailability, and is most amenable to conventional formulation development. The method of counterion determination needs to be precise, accurate, and rugged so that it easily can be transferred to other analytical laboratories where the active pharmaceutical ingredient is routinely monitored to ensure the safety, identity, strength, purity, and quality of the material. This material ultimately will be made into a drug product and consumed by the patient.

Several options exist for counterion determinations. The most commonly employed determination utilizes ion-exchange chromatography (IC), which was introduced in 1975 (1). In IC, conductivity detection is typically used and a suppressor is required to reduce the background signal. Over the last 30 years, IC with conductimetric detection has proven to be a very sensitive detector for both cations and anions. However, to perform a cation separation, for example, a cation exchange column with a cation suppressor is required to get adequate sensitivity. The same is true for anions, but utilizes an anion exchange column and suppressor. An alternative approach would employ strong anion or strong cation exchange columns in conjunction with UV detection for the determination of organic acids, or evaporative light scattering detection (ELSD) for detection of inorganic salts. Capillary electrophoresis (CE) also has been shown to be useful for counterion analysis and a method for simultaneous determination of anionic metabolites based upon CE–mass spectrometry (MS) has been shown to be specific and selective (2).