Quantitative Determination of Sodium-Octanoate in Human Serum Albumin Preparations

Requests for reprints to: Dr. T. Dengler, DRK-Blutspendezentrale Baden-Baden, Gunzenbachstraße 35, D-7570 BadenBaden (FRG) Introduction For many years commercially prepared human serum albumin (HSA) solutions for transfusion purposes has had to be heated for 10 h at 60 ¤C in the presence of stabilizers to inactivate transmissible infectious agents, e.g. hepatitis or human immune deficiency viruses (HIV). As thermal stabilizers, mainly sodium octanoate (sodium caprylate) and/or sodium acetyltryptophanate are used to avoid heat-denaturation of albumin [1–3]. For quality control of the final HSA product, in order to determine the sodium octanoate content, we use a simple and fast reversed-phase high-performance liquid chro-matography method, which we want to describe in this short report. Material and Methods Octanoic acid and decanoic acid were purchased from Merck (Darmstadt, FRG). Sample preparation: To 2 ml of the final 5 % or 20 % HSA solution an internal standard of 8 or 32mmol/l sodium decanoate from a stock solution was added and mixed well. 200 μl of this protein solution were added to 800 μl methanol and mixed with a vortex. The precipitate was centrifuged, and the supernatant was removed and filtered through a 0.45 μm Millex® HV filter (Millipore, Bedford, USA). Reversed-phase high-performance liquid chromatography of the extracts was done with a 4.6 × 250 mm Nucleosil®-C18 (5 μm) column (Machery-Nagel, Düren, FRG) with 0.1% trifluoroacetic acid (TFA) in methanol/water (80:20) as mobile phase at a flow rate of 0.8 ml/ min. 20 μl was injected. Detection was done by ultraviolet (UV) absorption at 214 nm. Each sample extract was injected at least twice and the peak areas were averaged. Results For reproduceable results we have established an internal standard reversed-phase HPLC method with sodium decanoate as standard substance. This standard, converted in the free acid form by an acid eluent, is well resolved from the octanoic acid and other small impurity peaks in the chromato-gram, shown in figure 1. For determination of the relative response at 214 nm and the relative recovery of octanoic and decanoic acid extracted from human serum albumin preparations, equal molar amounts CO CD λmL Λ