Abstract
A sensitive method for the separation and determination of
R(+)- and
S(−) enantiomers of pyridoglutehimide in serum by high performance liquid chromatography (HPLC) with UV detection was developed. The assay involves the use of a solid-phase extraction for serum sample clean-up prior to HPLC analysis using a C18 Bond–Elute column. Chromatographic resolution of the enantiomers was performed on a reversed-phase cellulose-based chiral column (Chiralcel OD-R, 250×4.6 mm I.D.) under isocratic conditions using a mobile phase of 25:75 v/v acetonitrile-0.3 M aqueous sodium perchlorate (pH 6.2 adjusted with perchloric acid) at a flow rate of 0.8 ml/min. Recoveries for
R(+)- and
S(−)-pyridoglutethimide enantiomers were in the range 86–91% at 300–900 ng/ml level. Intra-day and inter-day precision calculated as %R.S.D. were in the ranges of 2.9–3.9 and 1.5–4.7% for both enantiomers, respectively. Intra-day and inter-day accuracies calculated as percentage error were in the ranges of 1.9–3.3 and 1.5–3.9% for both enantiomers, respectively. Linear calibration curves in the concentration ranges of 100–1500 ng/ml for each enantiomer show correlation coefficient (
r) of more than 0.9995. The limit of quantification (LOQ) of each enantiomer was 100 ng/ml using 1 ml of serum. The detection limit (LOD) for each enantiomer in serum using a UV detection set at 257 nm was 50 ng/ml (
S/
N=2).