Owing to the lack of standards and certified reference materials for a variety of elemental species in specific matrices the application of species unspecific quantification strategies employing ICP-MS detection has gained more and more importance. The comparison of different calibration strategies by assessing their main sources of uncertainty is a prerequisite for accurate results. A flow injection (FI) model experiment, using a constant spike flow provided by a HPLC pump, and the chromatographic separation of three platinum species were conducted for fundamental evaluation of on-line isotope dilution mass spectrometry (IDMS) used for species unspecific quantification. A cisplatin standard solution was injected into a flow supplied by a HPLC pump for the FI study. This flow was mixed with a constant make-up flow containing an isotopically enriched 196Pt standard. A make-up (fsp) flow of 117 mg min−1 was determined gravimetrically. In comparison, a make-up flow calculation based on reverse ID calibration, i.e., injection of a standard solution of natural isotopic composition, was found to produce significantly biased results. Make-up flow values of 174, 148 and 143 mg min−1 were obtained depending on the peak width of the FI signal (0.7, 1.22, 1.39 min, respectively). A full combined uncertainty calculation, accounting for possible sources of uncertainty in the measurement process, was carried out for both methods and compared with an uncertainty budget which was set up for single point external calibration with internal standardization. On-line IDMS based on gravimetric spike mass flow determination showed a total combined uncertainty of 8%. A comparable uncertainty of 9% was calculated for single point external calibration with internal standardization, since with the investigated FI experiment sources of uncertainty such as gradient elution did not need to be considered. The high total combined uncertainty of 17% of the second investigated on-line IDMS procedure is mainly determined by mass-flow determination. Generally, the final combined uncertainty of on-line IDMS quantification was found to critically depend on the number of measured data points s−1 defining the peak. The species unspecific quantification methods were evaluated for HPLC-ICP-MS analysis of cisplatin and two of its hydrolysis products. On-line IDMS based on gravimetrical mass flow determination was an absolute prerequisite for obtaining accurate results in terms of total platinum concentration.