|dc.description.abstract||Clodronate, belonging to bisphosphonates, are used in treatment of various bone disease such as hypercalcemia of malignancy, Paget’s disease, osteolytic bone metastases and osteoporosis. In pharmacokinetic studies, many research have been reported for metabolism of clodronate in human biofluid such urine and plasma. However there is still a challenge for direct analysis of clodronate from complex system. Conventional method usually required precipitation and solid/liquid phase extraction for isolation followed by derivatization methods that are tedious and time-consuming.
We implemented our previously developed nanoprobe-based affinity mass spectrometry (NBAMS) assay for rapid extraction, detection and quantification of clodronate. Metal-chelating ligand, nitrilotriacetic acid (NTA), was covalently bonded on the MNPs to give NTA-PEG@MNPs and then immobilized with Ti4+ metal ion which provided high affinity for target molecules containing phosphonate group. Ti4+-NTA-PEG@MNP was demonstrated to serve as a high surface-to-volume ratio nanoprobe and applied to effective isolation of clodronate, a bone disease drug structural-containing phosphonate group, from human plasma. In the preliminary result, we have successfully demonstrated feasibility of NBAMS method for effective isolation of clodronate from human plasma. The detection of clodronate was achieved without any derivatization by using of MALDI-TOF MS with the LOD of 0.05 ng.
The major limitation in quantification by conventional MALDI MS is non-homogeneous crystallization on sample plate that results in poor signal reproducibility. With the introduction of seed-layer surface and spiked internal standard, we successfully reduced signal fluctuation from the improved homogeneous co-crystallization of analyte and matrix molecule. The calibration curve constructed by clodronate from human plasma has dynamic range from 200-8000 ng/ml with correlation coefficients better than 0.99. We demonstrated that a nanoprobe-based affinity mass spectrometry (NBAMS) is a simple, rapid, reproducible and accurate platform for simultaneous enrichment, detection and quantification of small molecular drugs in pharmaceutical metabolism study.