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An unexpected chromatographic behavior of fat globules has led to a simple HPLC assay for glycerin in the complex matrix of fat emulsions intended for intravenous injection. When a combination of a Hamilton PRP-1 (Polymer Reverse-phase) Column, a Biorad Microguard Carbo C Guard Column, and a Biorad HPLC Carbohydrate Analysis Column connected in series in the order listed was used for the separation of glycerin, with water as the mobile phase, the fat globules eluted unretained. The glycerin, which eluted by aqueous size exclusion, was detected and quantitated using a refractive index detector.

Safflower oil and soybean oil, which are the major constituents of fat emulsions for intravenous use, are both complex mixtures of triglycerides. Separation of triglycerides from the other ingredients of IV fat emulsion and their elution together as a single peak have been achieved through the use of a highly retentive Polymer Reverse Phase (PRP) column in tandem with a strong mobile phase. Quantitation is performed against a standard preparation of soybean oil using refractive index detection. The procedure is based on the use of the composite response from a known amount of one complex mixture of triglycerides to quantitate another complex mixture of triglycerides, which may even differ from the former in composition. A validation strategy for this unique situation has been developed and implemented.