A general approach for the quantitative analysis of bisphosphonates in human serum and urine by high‐performance liquid chromatography/tandem mass spectrometry

Abstract

Bisphosphonates are extremely hydrophilic and structurally similar to many endogenous phosphorylated compounds, making their selective extraction from serum or urine very challenging. Many bisphosphonates lack strong chromophores for sensitive UV or fluorescence detection. We report here the first general approach to enable sensitive and selective quantitation of N-containing bisphosphonates by liquid chromatography/tandem mass spectrometry (LC/MS/MS) following derivatization with diazomethane. The novelty of the strategy lies in performing the derivatization on silica-based anion-exchange sorbents as an integrated step in the sample purification by solid-phase extraction (SPE). The 'on-cartridge' reaction with diazomethane not only led to higher efficiency of derivatization, but also enabled a more discriminatory recovery of the drug's derivatives. The derivatized bisphosphonates demonstrated improved chromatographic separation and increased sensitivity of the detection. The general applicability of the approach was demonstrated by validation of bioanalytical methods for risedronate and alendronate in human serum and urine. Sensitivity was achieved at the pg/mL level with merely 100-200 microL of sample.

HPLC-MS/MS of Highly Polar Compounds

“…Di-and triesters resulted mainly from the derivatization of clodronate, as shown in figure 12; further optimisation is needed to increase the yield of one of the derivatives in order to use this approach for quantitative purposes. Zhu et al (2006) reported a methodology for the quantification of risedronate and alendronate by LC-MS/MS in biological samples after derivatization of the phosphonate groups with diazomethane. As a general approach, the urine or serum samples were loaded onto Bond-Elut SAX solid-phase extraction (SPE) columns that were washed with water prior to on-column derivatization with diazomethane.…”

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

“…Therefore, with recent improvement in the sensitivity of alendronate analytical assay, several research studies have attempted to optimize analysis of alendronate from plasma samples. A number of methods for the determination of alendronate in biological fluids have been published (Tsai et al, 1992;Usui et al, 1992Usui et al, , 1994Niemi et al, 1997;Sparidans and den Hartigh, 1999;Mönkkönen et al, 2000;Zhu et al, 2006;Tarcomnicu et al, 2007). Among these methods, high-performance liquid chromatography (HPLC) is a conventional technique that is readily used to detect alendronate in biological fluids.…”

“…However, these methods contain complex sample pretreatment processes which result in relatively low extraction efficiency and consequently, a large volume sample is necessary to detect trace amounts of alendronate. Recently, a sensitive liquid chromatography coupled with tandem mass spectrometry (LC/MS/MS) method is described to measure alendronate with only 500 µL of plasma regardless of extraction method (Zhu et al, 2006). However, high throughput analysis of alendronate using LC/MS/MS has some problems due to low ionization efficiency.…”

Chemometric-assisted determination of some bisphosphonates and their related substances in pharmaceutical forms by ion chromatography with inverse UV detection

“…Bisphosphonates have been analyzed in pharmaceutical forms [16,17] and human plasma [18,19] using UV spectrophotometry [20,21], RP-HPLC with UV detection [22,23], fluorimetric detection [24] ion chromatography [25], capillary electrophoresis [26,27], LC-MS [28] and LC-MS 2 [29].…”