Simultaneous quantification of D-penicillamine, D-penicillamine disulfide, and L-cysteine-D-penicillamine disulfide in human plasma: optimization of sample preparation and mass spectrometry procedures to support bioequivalence studies.

AIM: D-penicillamine (PSH) is an active pharmaceutical ingredient used for the treatment of various diseases, such as Wilson's disease, rheumatoid arthritis, cystinuria, and heavy metal poisoning. However, evaluating PSH in plasma poses substantial analytical challenges due to drug instability, thiol-disulfide exchange reactions with endogenous thiols, and the potential formation of multiple chemical forms of PSH. This study describes the development and validation of an analytical method to ensure the stability of PSH during analysis and enable accurate bioavailability measurements and bioequivalence assessments in human plasma.

METHODS

An ultra-high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) method was developed and validated to simultaneously determine PSH and its two major metabolites, D-penicillamine disulfide and L-cysteine-D-penicillamine disulfide, in human plasma. PSH stability was investigated under various experimental conditions to identify the optimal sample preparation procedure. The method was then applied to conduct a 90-day stability study of PSH in human plasma stored at -80°C.

RESULTS AND

CONCLUSION

A sensitive and specific analytical method was developed and validated in accordance with the US FDA M10 guidance. PSH remained stable under optimized conditions for at least 90 days. This method provides pharmaceutical researchers with a standardized approach for PSH pharmacokinetic analysis and bioequivalence evaluations.