Quantitation of iron oxide (Fe(2)O(3)) in pharmaceutical formulations using electrospray ionization mass spectrometry (ESI-MS) following iron complexation with 4-(2-pyridylazo)resorcinol (PAR) was evaluated. Complexation of Fe(III) with PAR was found to produce an intense mass spectral signal at low pH compared to the free ligand. Using the selected-ion-monitoring mode of a triple-quadrupole mass spectrometer, the signal at m/z 484 arising from the singly charged [Fe(III)(PAR)(2)](+) complex gave a limit of detection of ~2 µM for total iron. The linear range of the calibration curve was found to be 2-43 µM total iron. Trace iron interference was decreased to submicromolar levels by performing an optimized labware cleaning protocol and instrument replumbing using Polyetheretherketone (PEEK) tubing. The validation parameters of this ESI-MS total iron analysis (specificity, linearity, precision, accuracy, robustness and stability) are within the acceptance criteria listed in the validation guidelines developed by the Food and Drug Administration for industry. Recovery of 93% of the added iron indicated a satisfactory extraction procedure for tablets containing Fe(2)O(3) pigment. There was no statistical difference between the results obtained by ESI-MS and the common elemental method, inductively coupled plasma-optical emission spectroscopy. Since the proposed ESI-MS method can be performed on a mass spectrometer equipped with an ESI source, which is standard instrumentation in the pharmaceutical industry, the method validated here provides an alternate method for metal ion analysis by ESI-MS.