Effect of 18-Crown-6 on Oxytocin Stability in Aqueous Buffer Solutions

Abstract

In this study, the effect of 18-crown-6 on the stability of oxytocin in aqueous solution was explored. The study found that while 12-crown-4 and 15-crown-5 do not stabilize oxytocin, 18-crown-6 does have a stabilizing effect in citrate/phosphate buffer at pH 4.5. However, in acetate buffer at the same pH, the presence of 18-crown-6 had a destabilizing effect, possibly leading to a different degradation pathway. Both the stabilizing and destabilizing effects, depending on the buffer used, are concentration dependent where a higher concentration of 18-crown-6 is linked to a stronger effect. It is hypothesized that this effect may be linked to 18-crown-6 binding to the protonated ammonium group of oxytocin. Upon changing the mobile phase used in high-performance liquid chromatography experiments, we observed evidence supporting this binding hypothesis. When an acidic mobile phase was used (0.01% trifluoroacetic acid (TFA)), a partial shift in oxytocin retention time was observed for samples in acetate buffers in the presence of 18-crown-6 when using a 150 mm column (C18). The amount of the peak that shifted depended on the 18-crown-6 concentration used. A similar shift in oxytocin peak retention time was observed for samples in both acetate and citrate/phosphate buffers when using a 250 mm column (C18), but the peak completely shifted in those samples. When using an even more acidic mobile phase (0.1% TFA), the oxytocin peaks all had the same retention time again. Ultraviolet and nuclear magnetic resonance spectroscopy experiments also showed that the presence of 18-crown-6 has an observable effect on the resulting oxytocin spectra.

Figure 1

Hypothesized stabilizing interaction between 18-crown-6 (blue) and the protonated ammonium group (red) on the oxytocin molecule.

Figure 2

HPLC traces of oxytocin after 14 days of storage at 50 °C in the presence and absence of 2% w/v of 18-crown-6 in both acetate and citrate/phosphate buffer.

Figure 3

Ultraviolet (UV) trace from HPLC runs of an oxytocin standard (black), an oxytocin sample with 0.1% w/v of 18-crown-6 added (blue), and an oxytocin sample with 1.0% w/v of 18-crown-6 added (red). These samples were prepared in acetate buffer.

Figure 4

HPLC traces of samples showing an oxytocin peak shift when in the presence of 18-crown-6. The samples were prepared in citrate/phosphate buffer (left) and in acetate buffer (right). The mobile phase used was A: 0.01% TFA in H₂O and B: 0.01% TFA in 70% MeCN: 30% H₂O.

Figure 5

HPLC traces of samples showing no oxytocin peak shift regardless of the presence of 18-crown-6. The samples were prepared in citrate/phosphate buffer (left) and acetate buffer (right). The mobile phase used was A: 0.1% TFA in H₂O and B: 0.1% TFA in 70% MeCN: 30% H₂O.

Figure 6

UV absorption spectra for oxytocin, 18-crown-6, a solution with both oxytocin and 18-crown-6, and a solution with oxytocin, 18-crown-6, and potassium chloride.

Figure 7

Stacked NMR spectra of pure oxytocin (blue) and a mixture of oxytocin and 18-crown-6 (black) in D₂O.