Validation of a simple and sensitive gas chromatographic method for the analysis of tri-n-butyl phosphate from virally inactivated human immunoglobulin

Tri-n-butyl phosphate (TnBP), a solvent used in combination with Triton X-100 to inactivate lipid-enveloped viruses from immunoglobulin purified from human plasma is routinely measured in our laboratories by gas chromatography–flame ionization dectection (GC–FID) after extraction with C-18. We modified our present assay by extracting the analyte into hexane prior to measurement by GC–FID. We also found that the addition of a small volume of ethanol to the organic layer facilitates the extraction process by breaking the resulting emulsion formation caused by the hexane addition. The sample preparation and subsequent assay were fully validated in our laboratory. The process time for each sample is less than 2 min, a 15-fold improvement over solid-phase extraction techniques that were previously used in our laboratories. The recovery of TnBP in immunoglobulin using this newer method approximates 100%. The limit of quantitation (LOQ) was found to be 2 μg/ml or 2 ng per injection. The linear dynamic range of the assay is reported to be from the LOQ up to 50 μg/ml. The method is simple, relatively inexpensive and rapid. In addition, validation of the method demonstrates that it is accurate, precise, rugged and robust as demonstrated by reproducibility between analysts, instruments, laboratories, and columns. Finally, no problems were observed with regard to sample carryover.