Liquid chromatography-mass spectrometry analysis of five bisphosphonates in equine urine and plasma.

Bisphosphonates are used in the management of skeletal disorder in humans and horses, with tiludronic acid being the first licensed veterinary medicine in the treatment of lameness associated with degenerative joint disease. Bisphosphonates are prohibited in horseracing according to Article 6 of the International Agreement on Breeding, Racing and Wagering (published by the International Federation of Horseracing Authorities). In order to control the use of bisphosphonates in equine sports, an effective method to detect the use of bisphosphonates is required. Bisphosphonates are difficult-to-detect drugs due to their hydrophilic properties. The complexity of equine matrices also added to their extraction difficulties. This study describes a method for the simultaneous detection of five bisphosphonates, namely alendronic acid, clodronic acid, ibandronic acid, risedronic acid and tiludronic acid, in equine urine and plasma. Bisphosphonates were first isolated from the sample matrices by solid-phase extractions, followed by methylation with trimethylsilyldiazomethane prior to liquid chromatography - tandem mass spectrometry analysis using selective reaction monitoring in the positive electrospray ionization mode. The five bisphosphonates could be detected at low ppb levels in 0.5mL equine plasma or urine with acceptable precision, fast instrumental turnaround time, and negligible matrix interferences. The method has also been applied to the excretion study of tiludronic acid in plasma and urine collected from a horse having been administered a single dose of tiludronic acid. The applicability and effectiveness of the method was demonstrated by the successful detection and confirmation of the presence of tiludronic acid in an overseas equine urine sample. To our knowledge, this is the first reported method in the successful screening and confirmation of five amino- and non-amino bisphosphonates in equine biological samples.

A sensitive post-column photochemical derivatization/fluorimetric detection system for HPLC determination of bisphosphonates.

A new reversed-phase ion-pair high-performance liquid chromatographic (HPLC) method has been developed for the determination of the following bisphosphonic acids: alendronic acid (ALEN), etidronic acid (ETID), ibandronic acid (IBAN) and risedronic acid (RISE). Separation was achieved on a C(18) column using a mixture of 50 mmol L(-1) borate buffer pH 9.0 containing 0.25 mmol L(-1) tetrabutylammonium chloride and 0.5 mmol L(-1) EDTA and acetonitrile (97:3) as the mobile phase. The sensitive detection of the above bisphosphonic acids was based on their oxidation to orthophosphate by the on-line peroxydisulfate-assisted photolysis followed by post-column reaction with molybdate to yield phosphomolybdate. This subsequently reacted with thiamine to generate thiochrome and, finally, the fluorescence of thiochrome was measured at 440 nm with excitation at 375 nm. The developed method is precise with a mean relative standard deviation of 1.3%, sensitive (with a detection limit at the nmol L(-1) level), accurate, specific, rapid (analysis time approximately 13 min) and inexpensive because to the low cost of the reagents. The assay was applied to the analysis of the four bisphosphonic acids in commercial dosage formulations, in which the excipients did not interfere with the determination. The method was also applied to the determination of etidronate, risedronate and ibandronate in human urine. Sample preparation involves precipitation of the analytes from urine along with endogenous phosphates such as calcium salts by addition of calcium chloride at alkaline pH and dissolution of the precipitate in 0.05 mol L(-1) ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid.

[Separation of zoledronic acid and its related substances by ion-pair reversed-phase high performance liquid chromatography].

A rapid and simple ion-pair reversed-phase high performance liquid chromatographic method (HPLC) has been established for the routine analysis of zoledronic acid and its related substances. The chromatographic conditions were optimized based on the satisfactory separation of zoledronic acid from imidazol-1-ylacetic acid, their retention times and peak shape. The excellent separation of zoledronic acid from its related substances, including the remaining imidazol-1-ylacetic acid used in the synthesis of zoledronic acid and other impurities of oxidation and decomposition, was achieved within 9 min on a Hypersil C8 column with UV detection at 220 nm. The mobile phase was a mixture of methanol (20%) and 5 mmo/L phosphate buffer (80%) that contains 6 mmol/L tetrabutylammonium bromide. The resolution factor of zoledronic acid from its adjacent peak was more than 2.5. This is a simple and rapid method for the routine assay of zoledronic acid.

Bisphosphonates derived from fatty acids are potent inhibitors of Trypanosoma cruzi farnesyl pyrophosphate synthase.

Studies on the mode of action of a series of bisphosphonates derived from fatty acids, which had previously proved to be potent inhibitors against Trypanosoma cruzi proliferation in in vitro assays, have been performed. Some of these drugs proved to be potent inhibitors against the intracellular form of the parasite, exhibiting IC(50) values at the low micromolar level. As bisphosphonates are FDA clinically approved for treatment of bone resorption disorders, their potential innocuousness makes them good candidates to control tropical diseases.

Studies on the preparation and stability of samarium-153 propylene diamine tetramethylene phosphonate (PDTMP) complex as a bone seeker.

Propylene diamine tetra methylene phosphonate (PDTMP) was synthesised by modifying a method reported for the synthesis of EDTMP. Complexation of the synthesised phosphonate ligand with 153Sm was carried out by varying the experimental parameters and the complex was radiochemically characterized. Biodistribution studies showed that the uptake by bone in rats was 2% per g of bone, which was retained upto 48 h. The uptake by other organs was insignificant, except by the liver which showed a slightly higher absorption.

Preparation of (188Re) Re-AEDP and its biodistribution studies.

The synthesis of the Re (V) complex and preparation of 188Re-AEDP are described using 188Re which was obtained from the alumina-based 188W/188Re generator. Dependence of the radiolabeling yields of 188Re-AEDP on reducing agent concentration, AEDP concentration, pH and addition of carrier was examined. In the case of optimum conditions, the radiolabeling yields of 188Re-AEDP were 92-93% for carrier-free 188Re and 95-98% for carrier-added 188Re. The stability of 188Re-AEDP at pH approximately 6 was studied and it is found that the carrier has a significant effect on the stability of 188Re-AEDP. The biodistribution of carrier-free and carrier-added 188Re-labelled compounds in rats was also measured. The results show that 188Re (carrier-added)-AEDP is a potential bone palliation radiopharmaceutical due to its high skeletal uptake, rapid blood clearance and relatively low soft tissue absorption.

Effect of the composition of the eluent in the chromatography of 99mTc-diphosphonate complexes.

When the ligand and the reducing agent are added to the eluent used in the separation of 99mTc-diphosphonate complexes by column chromatography, a significant increase in recovery is obtained. The addition of the ligand and the reducing agent also influences the shape of the chromatogram.