The amount of synovial fluid in small joints which is available for analysis of endogenous compounds is usually very limited and saline washings are therefore commonly required. However, since the washing fluid is not fully recovered and, furthermore, dilutes the synovial fluid in the joint aspirate, the true synovial fluid concentration of a particular substance is unknown. In the present study we validate the use of vitamin B12 (hydroxocobalamin, 1 mg ml-1) as an internal standard to determine this dilution, since its strong red colour lends itself to spectroscopic measurement by a capillary tube system requiring only 3 microliter per sample. The absorbance of different dilutions of the stock solutions (25, 33 and 50% vitamin B12 in saline) by saline, plasma and hyaluronan was measured in order to establish the optimal wavelength, the lower limit of detection and the interference by plasma or hyaluronan in the absorbance. Furthermore, the interaction of vitamin B12 in the analyses of interleukin-1 beta, serotonin and glucose was investigated. The optimal wavelength was found to be 350 nm and the limit of detection 0.5-0.9% dilution of the vitamin B12 solution. Hyaluronan or plasma did not influence the absorbance measurements and the minute interaction of vitamin B12 in the photometric measurements could be compensated for.