Development and clinical application of a stability-indicating chromatography technique for the quantification of diazoxide

Diazoxide is a potential candidate for the treatment of transitional hypoglycaemia in infants. A clinical trial is currently underway to investigate whether low-dose oral diazoxide is beneficial for severe or recurrent transitional neonatal hypoglycaemia (the NeoGluCO Study, registration ANZCTR12620000129987). The present study aimed to develop and validate the parameters for quantifying diazoxide from neonatal plasma samples, and to assess the stability of extemporaneously prepared diazoxide suspensions to support the NeoGluCO Study. To determine the plasma concentration of diazoxide, a protein precipitation mediated extraction protocol was developed, which demonstrated >94% diazoxide extraction recoveries from all samples. The method was linear over the range of 0.2-40 mu g/mL (R-2 > 0.9994) with a limit of quantification of 0.2 mu g/mL. Accuracy of the method was within 97-106% with relative standard deviation < 6% for all samples. Diazoxide-plasma samples were stable for up to three months at-20 degrees C and up to 48 h when stored in the auto-sampler. Samples were stable for up to two freeze-thaw cycles, with further cycles compromising stability of diazoxide in plasma. The developed method was applied to determine chemical stability of the extemporaneously prepared diazoxide suspensions. These were stable at both 2-8 degrees C and 25 degrees C/60% RH, with 98% of diazoxide remaining after 35 days in both storage conditions. Diazoxide was successfully quantified from plasma collected from six neonates enrolled in the NeoGluCO Study, using the developed protocol. Overall, an efficient and reproducible extraction protocol was developed and validated for the estimation of diazoxide from human plasma.

Automated summary

The present study developed and validated a efficient extraction method for quantifying diazoxide from neonatal plasma. The method showed good linearity and accuracy, and demonstrated the stability of diazoxide in plasma.