(M0930-06-32) Method for the Analysis and Quantitation of Pharmaceutical Counterions Utilizing Hydrophilic Interaction Liquid Chromatography

Purpose: More than 50% of drugs manufactured today are produced in the drug salt form. Active Pharmaceutical Ingredients (APIs) are attached to salts increase the physiochemical properties and bioavailability of drugs. The identification and quantification of API freebase and salt counterions is crucial for quality control (QC) testing in the pharmaceutical industry. Typically, QC testing for drug salts involves multiple methods. Zwitterionic Hydrophilic Interaction Liquid Chromatography (Z-HILIC) in combination with Evaporative Light Scattering Detection (ELSD) provide a unique solution to these QC testing challenges. Here we developed a single LC method to separate and quantify both pharmaceutical cations and anions from the API free base in drug substances.

Methods: Chromatographic separation was performed using an ACQUITY^™ Arc™ Premier LC System utilizing a Waters™ 2424 Evaporative Light Scattering Detector equipping an Atlantis™ Premier BEH™ Z-HILIC Column. The Column was heated to 40 °C, flow rate set to 1.4ml/min, and a 10μL sample injection volume. The method utilized a four mobile phase gradient. The gradient pumped constant concentrations of 0.5% formic acid and 10mM ammonium formate from stock mobile phase bottles. Further the amount of Acetonitrile decreased from 80% to 10% and is replaced by Deionized Water over the course of the run time (10 minutes). ELSD parameters were the following: detector gain of 75, gas pressure of 40psi, and drift tube temperature of 50°C. The nebulizer heating/cooling function was disabled. Raw data was processed and calculated using Waters Empower™ 3 Software Build 3471.

Results: The HILIC / ELSD method demonstrated reproducible separation of Pharmaceutical counterions. Over the course of ten injections of a mixed counterions standard, %RSD for area and retention time were 6% and 5%, respectively. The counterions standard was used as an ion identification tool for drug substance samples, using the retention time. Further, this method provided dynamic linear ranges for three commonly used salts: Potassium, sodium, and chloride. Based upon the solubility of each salt and detector capacity, the linear ranges were individually calculated for each counterion. Potassium, sodium, and chloride were linear between 8mM to 40mM, 4mM to 60mM, and 2mM to 20mM, respectively. The R² for all curves was >0.997 using a linear log/log fit as recommended by the owner’s manual. These curves were used to quantitate the amount of salt in pharmaceutical drug salt formulations. Naproxen sodium, metformin hydrochloride, and losartan potassium were quantified against the sodium, chloride, and potassium curves, respectively. The sodium, chloride, and potassium ions in the drug salts were quantitated at 16.4mM, 2.79mM, and 2.96mM respectively. Further, the API free bases of these drugs salts were separated from the pharmaceutical counterions. Some API free bases, such as metformin, had considerable retention using this method.

Conclusion: Here we demonstrated a single LC method for the separation and quantitation of pharmaceutical counterions from free base APIs.

Acknowledgements: ACQUITY, Arc, Waters, and Empower are trademarks of Waters Corporation.


Graphic 1A. Overlay of 10 Chromatograms for the counterions standard mixture.


Graphic 2A. The calibration curve for Sodium spanning from the concentrations of 4mM to 60mM.
Graphic 2B. The calibration curve for Chloride spanning from the concentrations of 2mM to 20mM.
Graphic 2C. The calibration curve for Potassium spanning from the concentrations of 8mM to 40mM.


Graphic 3A. An ELSD Chromatogram of the counterions standard (black). The specific peaks in the chromatogram are Nitrate (1), Potassium (2), Sodium (3), Chloride (4), Phosphate (5), Magnesium (6), and Calcium (7). This is used as a known counterions reference to be compared to the drug samples in Graphic 3B, 3C, and 3D. The known counterions are designated by an arrow that extends from Graphic 3A to the respective ion in the drug samples (Graphic 3B, 3C, and 3D).
Graphic 3B. An ELSD chromatogram of the drug Naproxen Sodium prepared at 3mg/mL (red). The Naproxen free base is visible at approximately 0.6 minutes and the sodium ion is visible at approximately 3.1 minutes. This sodium peak lines up with the known sodium peak in Graphic 3A. The sodium ion was quantified at 16.397mM.
Graphic 3C. An ELSD chromatogram of the drug Metformin Hydrochloride prepared at 0.5mg/mL (orange). The Metformin free base is visible at approximately 1.7 minutes and the chloride ion is visible at approximately 3.4 minutes. This chloride peak lines up with the known chloride peak in Graphic 3A. The chloride ion was quantified at 2.788mM.
Graphic 3D. An ELSD chromatogram of the drug Losartan Potassium prepared at 10mg/mL (blue). The Losartan free base is visible at approximately 0.7 minutes and the potassium ion is visible at approximately 3.0 minutes. This potassium peak lines up with the known potassium peak in Graphic 3A. The potassium ion was quantified at 21.205mM.