Development and validation of a reverse phase HPLC method for the determination of caprylic acid in formulations of therapeutic immunoglobulins and its application to antivenom production.

A novel method of high performance liquid chromatography with UV detection for the quantification of caprylic acid in formulations of therapeutic immunoglobulins was developed and validated. Samples have interfering proteins that were removed by ultrafiltration in a centrifugal filter unit of 10 kDa nominal molecular weight limit. Then, compounds present in ultrafiltrates were separated on an Eclipse XDB-C8 5 microm column (150 mm x 4.6 mm i.d.), using a mixture of acetonitrile-water (60:40, v/v) as the mobile phase at a flow rate of 1 mL/min. The UV detection was performed at 210 nm. The method was found to be precise and accurate, with a linearity range from 400 microg/mL to 600 microg/mL (r=0.9948). The limit of detection (LOD) and the limit of quantification (LOQ) were found to be 13.46 microg/mL and 44.85 microg/mL, respectively. To illustrate the usefulness of the method in the in-process and final quality control for production of therapeutic immunoglobulin formulations, permeates obtained from the industrial diafiltration step in the manufacture of equine-derived snake antivenoms and ten batches of finished product were analyzed.