A new chromatographic approach for separating cacao procyanidins according to their degree of polymerization has been developed. It utilizes diol stationary phase columns operating in normal phase mode with a binary gradient of acidified acetonitrile and methanol-water. Performance of the diol stationary phase was evaluated on an analytical scale utilizing classical chromatographic conditions for the normal phase separation of procyanidins according to their degree of polymerization. The new separation approach was developed on an analytical scale but further extended to the preparative scale. These newly developed analytical and preparative high-performance liquid chromatography procedures were successfully applied to the separation, as well as isolation, of cacao procyanidins from unfermented cacao seeds. The degree of polymerization associated with each molecular weight fraction was determined by mass spectrometry.

A sensitive and precise LC-ESI-MS/MS method for determination of vandetanib (ZD6474) in human plasma and cerebrospinal fluid (CSF) using d4-ZD6474 as an internal standard (ISTD) was developed and validated. Sample preparation consisted of a simple liquid-liquid extraction with tert-butyl methyl ether containing 0.1% or 0.5% ammonium hydroxide. ZD6474 and ISTD were separated on a Kinetex C18 column (2.6 μ, 50 mm × 2.1 mm) at ambient temperature with an isocratic mobile phase (acetonitrile/10mM ammonium formate = 50/50, v/v, at pH 5.0) delivered at 0.11 mL/min. The retention time of both compounds was at 1.60 min in a runtime of three min. Detection was achieved by an API-3200 LC-MS/MS system, monitoring m/z 475.1/112.1 and m/z 479.1/116.2 for vandetanib and ISTD, respectively. The method was linear in the range of 0.25 to 50 ng/mL (R2 ≥ 0.990) for the CSF curve and from 1.0 to 3,000 ng/mL (R2 ≥ 0.992) for the plasma curve. The mean recovery for vandetanib was 80%. Within-day and between-day precisions were ≤ 8.8% and ≤ 5.9% for CSF and plasma, respectively. Within-day and between-day accuracies ranged from 95.0 to 98.5% for CSF, and from 104.0 to 108.5% for plasma. Analysis of plasma from six different sources showed no matrix effect for vandetanib (MF = 0.98, %CV ≤ 4.97, n = 6). This method was successfully applied to the analysis of pharmacokinetic samples from children with brain tumors treated with oral vandetanib.