Solid-phase extraction and validated spectrofluorimetric quantification of pamidronate in human plasma

Purpose: To design a simple and sensitive quantification procedure for pamidronate disodium (PAM) after its solid phase extraction from plasma.Methods: The procedure was based on derivatization of PAM using a suitable fluorogen, 4-chloro-7-nitro-2,1,3-benzoxazole. The product was determined spectrofluorometrically at excitation and emission wavelengths of 390 and 535 nm, respectively. The method was optimized for all factors that affect the reaction between PAM and the fluorogen. These factors include diluting solvent, pH of the reaction medium, volume of fluorogen solution, buffer pH, buffer volume, temperature and heating time. The method was fully validated according to US-FDA guidelines with respect to linearity, accuracy, precision, recovery, robustness and stability.Results: PAM was successfully extracted from human plasma with solid-phase extraction technique. A linear response was obtained in the concentration range of 10 – 100 ng/mL, with correlation coefficient of 0.998. Mean maximum plasma concentration of PAM was 9.73.2 ± 3.2 µmol/L, which was within the linear range of the proposed method, thereby confirming its sensitivity for the determination of plasma PAM.Conclusion: The proposed procedure is suitable for the quantification of PAM in human plasma after its solid phase extraction. The method is sensitive enough for use in PAM determination in pharmacokinetic studies. Moreover, it is likely a more cost-effective and simpler alternative method than high performance liquid chromatograph (HPLC) methods. Keywords: Pamidronate disodium, Derivatization, Spectrofluorimetry, Fluorogen, Quantification