Assay for D-penicillamine-protein conjugate in human plasma utilising chemical reduction followed by high-performance liquid chromatography with gold/mercury electrochemical detection.

D-Penicillamine (D-pen), a thiol with antirheumatic activity, forms a mixed disulphide with albumin in vivo. This conjugate is important in the pharmacokinetics and possibly the mode of action of D-pen. An assay was devised for D-pen-albumin disulphide, based on separation from plasma by acid precipitation followed by quantitative reduction with sodium borohydride in an anoxic environment. The liberated D-pen was then assayed by high-performance liquid chromatography with gold/mercury electrochemical detection. The assay was sensitive to 1.2-microM D-pen-albumin disulphide (signal-to-noise ratio greater than 2), absolute recovery was 92.7% and intra-assay coefficient of variation was 4.6% in human plasma. This technique also may be useful for quantitating protein conjugates of other thiols.