Detection of minimal calcium amounts in acids possessing erosive potential

Objective: The study evaluates the feasibility of the Arsenazo III method to detect minimal amounts of calcium in small volumes of acids known to possess erosive potential in dental hard tissue. Methods: Hydrochloric acid, oxalic acid, maleic acid, phosphoric acid, tartaric acid, citric acid, lactic acid and acetic acid were adjusted to pH 2.0, 2.3 and 3.0. Small amounts of calcium 124 pmol/10µl, 247 pmol/10µl, 371 pmol/10µl or 494 pmol/10µl were dissolved in 10µl of the respective acid, placed into wells of a microplate reader and analyzed for calcium. A mixture of the calcium concentrations with distilled water served as control. The experiments were run with ten repeats in series. For assessing reproducibility and precision of the technique, intra- and inter-assay coefficient of variation (CV) was calculated. Additionally, lower limit of quantification (LLOQ) was determined. Results: Intra-assay-CVs amounted to <10% for most of the acidic solutions at all pH except for citric acid (pH: 2.0, 2.3, 3.0), acetic acid (pH: 2.0), phosphoric acid (pH: 3.0) and tartaric acid (pH: 2.3). In most of the cases, intra-assay-CV distinctly decreased with increasing calcium concentration of the respective acid and showed higher values at lower pH. At pH 2.3 and 3.0, the solutions with 371 pmol/well and 494 pmol/well showed an intra-assay-CV <3%, except for citric acid and phosphoric acid. The inter-assay-CV was below 20% for all tested acidic solutions except for measuring lowest calcium concentration (123 pmol/well) in citric acid at pH 2.0 and 2.3, and for measuring 247 pmol/well at pH 2.3. Depending on the acid, LLOQ showed a range from 23.8 pmol/well (phosphoric acid: pH 2) to 190.5 pmol/well (citric acid: pH 2.3). Conclusions: Depending on the acid used, the Arsenazo III method is a reliable tool to detect and quantify minimal calcium contents in small volumes of acidic solutions.