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.
Joyce
D A
DA
University of New South Wales, School of Physiology and Pharmacology, Australia.
Wade
D N
DN
D001172:Arthritis, Rheumatoid / Q000097:blood; D001798:Blood Proteins / Q000378:metabolism*; D001894:Borohydrides; D002851:Chromatography, High Pressure Liquid; D004220:Disulfides / Q000032:analysis; D004563:Electrochemistry; D006046:Gold / Q000032:analysis; D006801:Humans; D008628:Mercury / Q000032:analysis; D010084:Oxidation-Reduction; D010396:Penicillamine / Q000097:blood*; D011485:Protein Binding
D001798:Blood Proteins; D001894:Borohydrides; D004220:Disulfides; D006046:Gold; D008628:Mercury; D010396:Penicillamine