The influence of sample temperature on water cluster ion formation for ToF-SIMS studies of frozen hydrated samples

In time-of-flight secondary ion mass spectrometry (ToF-SIMS), analysis of frozen biological specimens at cryogenic temperatures is often necessary to maintain the native 3D structure of the specimen. Frozen hydrated analysis results in interferences from sputtered water cluster peaks that extend over the full spectral mass range. In this study, we have investigated the influence of the analysis temperature from 98 to 183 K on the water cluster spectrum from a frozen hydrated cell-free model biofilm system which contained the antibiotic ciprofloxacin. Below 163 K, the spectrum was dominated by sequences of water cluster ions of the form (H₂O)_nX⁺, where X⁺ is either H⁺, NH₄⁺, or one of at least 16 other small cations. These sequences repeat every 18 mass units. These sequences of water cluster ions begin at m/z 19 and extend to over m/z 2000. Different temperature trends were observed for each cationized water cluster sequence. At a temperature of 153 K, just below the onset of freeze-drying, the (H₂O)H⁺ cluster signals decline, and many cationized cluster signals go through a local minimum. In this same temperature region, an increase in proton mobility was observed in experiments using D₂O. The decline in water cluster ion signals at 153 K was accompanied by an increase in the [ciprofloxacin+H]⁺ signal as well as an increase in signals from other organic molecules. Based on these results, 153 K is recommended as the optimum temperature for analysis of ciprofloxacin in frozen hydrated specimens.