Frequently, requests come to Xendo Drug Development to support clinical trials involving bisphosphonates, indicated for the prevention or treatment of postmenopausal and glucocorticoid-induced osteoporosis and Paget’s disease. Usually, urine is used to assess pharmacokinetics, since clearance from plasma occurs very rapidly. Bioanalysis of bisphosphonates is one of the major challenges of today’s bioanalytical scene, as a result of a complex of factors: pharmacokinetic and pharmacological properties, chemical properties and matrix issues.

After a long period of bioanalytical research activities, in which a wide range of assay principles and sample preparation modes have been investigated and have failed, Xendo has succeeded in developing an ultrasensitive, rapid and very specific methodology for bisphosphonates in biological samples. A detailed description of our approach is outlined below. Please feel free to contact us in case you are interested in our bisphosphonates technology.

Literature for sensitive bioanalytical methods for bisphosphonates is scarce, so the development of a generic method for bisphosphonates appeared to be one of the major challenges for the laboratory so far and asked a lot of the skills of the people involved. The major challenge in bioanalysis of bisphosphonates is given by their extreme polarity and their lack of bioanalytical assay sensitivity, which makes them unsuitable for direct gas chromatographic LC-MS/MS or even LC-ICP-MS analysis (though we tried). Although risedronate is one of the few bisphosphonates that possesses a chromophore (pyridine group), suitable for ultra-violet detection, it was observed that the sensitivity was not sufficient by far for pharmacokinetic analysis in urine and plasma.

To make bisphosphonates (more) accessible for analysis, they are usually analyzed after derivatization of the primary or secondary amine group present in the molecule. Even then however, the sensitivity is hardly sufficient for analysis in urine and insufficient for analysis in plasma. On top of that, risedronate contains a tertiary amine which makes the molecule unsuitable for derivatization with amine reacting agents.

Besides the poor sensitivity another major challenge was the clean-up of the samples due to their mentioned chemical nature. Sample clean-up prior to analysis is mandatory to avoid blocking of columns and to prevent severe ion-suppression in the MS/MS system.

To overcome all the above-mentioned difficulties, Xendo searched for an answer and came up with an elegant and highly sensitive method that appeared to be suitable for determination in plasma as well, without employing tedious and ‘old-fashioned’ repeated calcium precipitation.

The method developed by Xendo is based on simultaneous sample clean-up and on-column derivatization of the phosphate groups into a derivative that can be readily analyzed on a LC-MS/MS system. Due to the uniformity of the procedure, this method is also suitable for other bisphosphonates (e.g. alendronate, pamidronate, etidronate). The high sensitivity of the developed method also enables pharmacokinetic analysis in serum or plasma.