A New Liquid Chromatography—Mass Spectrometry-Based Method to Quantitate Exogenous Recombinant Transferrin in Cerebrospinal Fluid: A Potential Approach for Pharmacokinetic Studies of Transferrin-Based Therapeutics in the Central Nervous Systems

Transferrin (Tf) is an 80 kDa iron-binding protein that is viewed as a promising drug carrier to target the central nervous system as a result of its ability to penetrate the blood–brain barrier. Among the many challenges during the development of Tf-based therapeutics, the sensitive and accurate quantitation of the administered Tf in cerebrospinal fluid (CSF) remains particularly difficult because of the presence of abundant endogenous Tf. Herein, we describe the development of a new liquid chromatography–mass spectrometry-based method for the sensitive and accurate quantitation of exogenous recombinant human Tf in rat CSF. By taking advantage of a His-tag present in recombinant Tf and applying Ni affinity purification, the exogenous human serum Tf can be greatly enriched from rat CSF, despite the presence of the abundant endogenous protein. Additionally, we applied a newly developed ¹⁸O-labeling technique that can generate internal standards at the protein level, which greatly improved the accuracy and robustness of quantitation. The developed method was investigated for linearity, accuracy, precision, and lower limit of quantitation, all of which met the commonly accepted criteria for bioanalytical method validation.