A validated LC method for the determination of vesamicol enantiomers in human plasma using vancomycin chiral stationary phase and solid phase extraction

An enantioseparation high performance liquid chromatographic (HPLC) method was developed and validated to determine D-(+)- and L-(-)-vesamicol in human plasma. The assay involved the use of a solid phase extraction for plasma sample clean up prior to HPLC analysis utilizing a C18 Bond-Elute column. Chromatographic resolution of the vesamicol enantiomers was performed on a vancomycin macrocyclic antibiotic chiral stationary phase (CSP) known as Chirobiotic V with a polar ionic mobile phase (PIM) consisting of methanol:glacial acetic acid:triethylamine (100:0.1:0.05 (v/v/v)) at a flow rate of 1.0 ml/min and UV detection set at 262 nm. All analyses were conducted at ambient temperature. The method was validated over the range of 1-20 microg/ml for each enantiomer concentration (R2>0.999). Recoveries for D-(+)- and L-(-)-vesamicol enantiomers were in the ranges of 96-105% at 3-16 microg/ml level. The method proved to be precise (within-run precision ranged from 1.3 to 2.7% and between-run precision ranged from 1.5 to 3.4%) and accurate (within-run accuracies ranged from 0.8 to 3.4% and between-run accuracies ranged from 1.7 to 5.0%). The limit of quantitation (LOQ) and limit of detection (LOD) for each enantiomer in human plasma were 1.0 and 0.5 microg/ml (S/N=3), respectively.