Factors affecting the analysis of 18O-enriched aqueous samples when using CO2 equilibration in vacutainers™

Increasing use of ¹⁸O-enriched water in biological studies such as those involving doubly labeled water has led to a requirement for a simple cost effective method for analyzing large numbers of ¹⁸O-enriched biological samples. We evaluated the influence of various factors on the precision and accuracy of an equilibration technique in which aqueous samples are equilibrated with CO₂ in evacuated blood collection tubes, Vacutainers^™. Using 10 mL Vacutainers with degassed rubber septa, 1 mL water samples equilibrated in an atmosphere of CO₂ after 16 h on an orbital shaker. δ¹⁸O values became more variable when the sample volume was reduced to 0.25 mL. Varying the evacuation time for the tubes prior to filling with CO₂ between 15 and 75 s had no marked influence on the results. Similarly, sonicating the water samples during this process to remove dissolved gases also had little effect. Bubbling air or CO₂ through the samples prior to analysis led to a reduction in the ¹⁸O content of enriched water samples due to the introduction of ¹⁶O; however, N₂ had no effect on the δ¹⁸O values. Memory effects between samples and instrumental drift were detected with large changes in enrichment (up to 140‰) during mass spectrometric analysis. An analytical procedure and calculations are presented, which in conjunction with two reference waters, may be used to remove these drift effects. This procedure coupled with suggested sample analysis sequences minimizes the influence of memory and allows precision to be optimized. Even in the absence of these precautions, analytical precision on water with δ¹⁸O_V-SMOW content of +30‰ was ca ±0.1‰, and at +175‰ was ±0.37‰, comparable to other ¹⁸O equilibration methods.