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.