Background  High‐quality methods for lipoprotein characterization are warranted in studies on various metabolic diseases.

Materials and methods  An automated system for size‐exclusion chromatography (SEC) of lipoproteins using commercially available components is described. Cholesterol or triglyceride content in separated lipoproteins from plasma and interstitial fluid (IF) was continuously determined on‐line using microlitre sample volumes.

Results  The lipoprotein assay showed a good concordance with the classic ultra‐centrifugation/precipitation technique using fresh or frozen samples. Determination of lipoproteins in IF obtained from vacuum‐induced skin blisters from 18 healthy subjects revealed that very low density lipoprotein (VLDL), low density lipoprotein (LDL) and high density lipoprotein (HDL) cholesterol levels were 18%, 19% and 25%, respectively, of concomitant plasma concentrations. The size‐exclusion chromatography (SEC) system also allows for triglyceride determination on‐line and it could be shown that the system is advantageous for an accurate determination of triglycerides in conditions when there are high levels of glycerol, e.g. in mice and in patients with hyperglycerolaemia (pseudo‐hypertriglyceridaemia).

Conclusions  The described system should be of value in studies where detailed lipoprotein analysis is warranted and particularly when significant sample series with small volumes are available. Our data also suggest that there is a 4–5·5‐fold concentration gradient between plasma and IF for the three major plasma lipoproteins.