Vacuoles play major roles in the trafficking, storage, and homeostasis of metal ions in fungi and plants. In this study, 29 batches of vacuoles were isolated from Saccharomyces cerevisiae. Flow-through solutions (FTS) obtained by passing vacuolar extracts through a 10 kDa cut-off membrane were characterized for metal content using an anaerobic liquid chromatography system interfaced to an online ICP-MS. Nearly all iron, zinc, and manganese ions in these solutions were present as low-molecular-mass (LMM) complexes. Metal-detected peaks with masses between 500-1700 Da dominated; phosphorus-detected peaks generally comigrated. The distribution of metal:polyphosphate complexes was dominated by particular chain-lengths rather than a broad binomial distribution. Similarly treated synthetic FeIII polyphosphate complexes showed similar peaks. Treatment with a phosphatase disrupted the LMM metal-bound species in vacuolar FTSs. These results indicated metal:polyphosphate complexes 6-20 phosphate units in length and coordinated by 1-3 metals on average per chain. The speciation of iron in FTSs from iron-deficient cells was qualitatively similar, but intensities were lower. Under healthy conditions, nearly all copper ions in vacuolar FTSs were present as 1-2 species with masses between 4800-7800 Da. The absence of these high-mass peaks in vacuolar FTS from cup1Δ cells suggests that they were due to metallothionein, Cup1. Disrupting copper homeostasis increased the amount of LMM copper:polyphosphate complexes in vacuoles (masses between 1500-1700 Da). Potentially dangerous LMM copper species in the cytosol of metallothionein-deficient cells may traffic into vacuoles for sequestration and detoxification.