Determination of pamidronate in human whole blood and urine by reversed‐phase HPLC with fluorescence detection

Abstract

Pamidronate is a bisphosphonate that is effective in treating bone disease including osteopenia and osteoporosis in adults. A sensitive and reliable method for the analysis of pamidronate in whole blood and urine is key to the development of this drug for use in children. A previously described method for pamidronate analysis serum and urine did not consistently detect the drug at satisfactory levels in whole blood. The procedure involves co-precipitation of the bisphosphonates with calcium phosphate, pre-column derivitization with fluorescamine, HPLC utilizing a Nucleosil C(18) column, and fluorescence detection with excitation at 395 nm and emission at 480 nm. Changes to the original protocol included the use of a new internal standard (alendronate), the optimization of the concentration of ethylenediaminetetraacetic acid (EDTA) for dissolving the precipitate, and the elimination of the acidification step prior to deproteinization. The optimum EDTA concentration, which had a significant effect on the labeling capability of fluorescamine, was determined to be 20 mm.A good separation between pamidronate and alendronate was achieved using a heated (40 degrees C ) Nucleosil C(18), 10 micro m particle size column. The mobile phase was an aqueous solution of 1 mm Na(2)EDTA-methanol (97:3, v/v) adjusted to pH 6.5 using a fl ow-rate of 1 mL/min. Fluorescence detection was set at 395 nm for excitation and at 480 nm for emission. The limit of quantitation for pamidronate was 0.5 micro g/mL in whole blood and 0.1 micro g/mL in urine. The method was applied to both whole blood and urine samples from pediatric patients.

Determination of alendronate in low volumes of plasma by column-switching high-performance liquid chromatography method and its application to pharmacokinetic studies in human plasma

“…Initially in this study, the reactions between alendronate and fluorescence dyes such as FMOC, fluorescamine and OPA was carried out according to methods established in previous papers (Ptáček et al, 2002;Al Deeb et al, 2004;Wong et al, 2004). Once formed, the FMOC-alendronate product was found to be stable for up to one month at 4 o C and showed high sensitivity.…”

“…FMOC (Ptáč ek et al, 2002), fluorescamine (Wong et al, 2004), or ophthalaldehyde (OPA) (Al Deeb et al, 2004) has been used in most of the previous studies for derivatization of amino-bisphosphonates.…”

HPLC-MS/MS of Highly Polar Compounds

“…Fluorescamine was one of the most used derivatization reagents of the amino group (Flesh et al, 1991, Wong et al, 2004. Aminoalkylphosphonic acids were isolated from urine or plasma by coprecipitation with calcium phosphate; then the precipitate was dissolved in Na 2 EDTA pH10 and reacted with fluorescamine.…”

Determination of zoledronic acid in human urine and blood plasma using liquid chromatography/electrospray mass spectrometry

“…For purification, co-precipitation with calcium phosphate under alkaline conditions [6][7][8] and subsequent removal of calcium ions using various solid-phase extraction columns [9,10] was the most frequently discussed approach. Unfortunately, due to the chemical nature of zoledronic acid, its chromatographic separation is challenging.…”