HPLC Analysis of Bucillamine by Derivatization with N‐(1‐Pyrenyl)Maleimide in Human Blood

Abstract

Bucillamine, N‐(2‐mercapto‐2‐methylpropionyl)‐L‐cysteine, is a novel disease‐modifying anti‐rheumatic drug containing two sulfhydryl groups. We investigated high performance liquid chromatographic (HPLC) analysis of bucillamine by derivatization with N‐(1‐pyrenyl)maleimide (NPM) in human blood. Human blood samples were immediately mixed with NPM at room temperature for 15 min and injected into an HPLC. The linearity was displayed for bucillamine concentrations ranging from 3 to 3000 nM (r = 0.999). The lower limit of detection of bucillamine derivative was 2.5 nM. Bucillamine derivative remained stable at −4°C for at least 2 weeks with less than 5% variation. The coefficients of variation for intra‐day and inter‐day assays were less than 4.2% and 5.3%, respectively. No endogenous and exogenous compounds (L‐cysteine, glutathione, N‐acetyl‐L‐cysteine, captopril, and D‐penicillamine), possessing a sulfhydryl group in these structures, were found to interfere with the peak of the bucillamine derivative. These results indicate that HPLC assay of bucillamine by derivatization with NPM is rapid, sensitive, and reproducible for determining bucillamine in human blood. This method may be applied to pharmacokinetic studies of bucillamine in humans.

Keywords:

• HPLC
• Bucillamine
• N‐(1‐pyrenyl)maleimide
• Derivatization

Acknowledgment

The authors thank Santen Pharmaceutical Co., Ltd. for the supply of bucillamine.