Isoflurane held in a sealed glass reservoir was plumbed to an <i>in vitro</i> tissue bath that was open to the atmosphere.

<p>A. In a representative experiment, measurements from the reservoir with ACSF saturated with isoflurane (diamond, 13.4 mM) or diluted; 1∶1 (triangle, nominal concentration 6.7 mM); 1∶3 (square, nominal concentration 3.35 mM) and 1∶9 (circle, nominal concentration 1.3 mM) were perfused through the bath and sampled at the bath outlet over time. There was a concentration dependent loss of isoflurane from the bath. Yet, isoflurane concentration in the tissue bath reached a steady state after ∼2 min (bath volume = 0.5 ml, flow rate 1.5–2 ml/min) B. Isoflurane cleared the bath within minutes when washed with ACSF at concentrations typically tested during <i>in vitro</i> electrophysiological studies.</p

The concentration of isoflurane in the bath, as measured by GC/MS, is plotted against the nominal concentration of isoflurane achieved by dilution of ACSF saturated with isoflurane.

<p>For measured values, each point represents the average of two samples taken during each isoflurane exposure (at 2 and 5 min, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0003372#pone-0003372-g004" target="_blank">Figure 4</a>). The range of values (grey shading) at each nominal concentration indicates the variability of bath isoflurane concentrations. This loss occurred consistently despite identical procedures for diluting isoflurane to nominal concentrations for each experiment.</p

Isoflurane was reliably detected and measured by GC/MS.

<p>Bath samples (100 µl) were taken and isoflurane was extracted into a heptane/halothane solution, where halothane served as an internal control. A. A representative chromatogram demonstrating the detection of isoflurane and halothane at typical retention times. The area ratio of each peak was compared and allowed for precise determination of isoflurane concentration. On each experimental day bath samples were processed along side a set of standards. B. The average of 21 standard calibration curves used during <i>in vitro</i> experiments (values = mean±SEM). Standard isoflurane concentrations and the measured area ratio of isoflurane to halothane were highly correlated (R² = 0.99). Insert; the greatest error between the curves occurred at 1000 µM isoflurane where the relative standard deviation (RSD) was 1.3%. The degree of accuracy in determining bath isoflurane concentration across experiments by GC/MS was very high.</p