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

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.

Peptidomic analysis of the skin secretions of the pickerel frog Rana palustris identifies six novel families of structurally-related peptides.

Peptidomics methodology has been used to identify and characterize structurally 26 previously undescribed peptides in the electrically stimulated skin secretions of the North American pickerel frog Rana palustris. Peptides in the secretions were analyzed by electrospray mass spectrometry and components in mass range 700-2500 Da, present in major abundance, were purified by reverse-phase HPLC. Cysteine-containing components were identified by treatment with dithiothreitol and 4-vinylpyridine and re-analysis of the derivatizated peptide by mass spectrometry. Application of these techniques led to the identification of six families of structurally-related peptides comprising (a). six peptides containing the consensus sequence Cys-Trp-Xaa-Thr-Lys-Ser-Ile-Pro-Pro-Lys/Arg-Xaa-Cys, (b). three peptides containing the consensus sequence Pro-Pro-Gly-Val-Cys-(Xaa)(3)-Lys/Arg-Arg-Cys, (c). two peptides containing the consensus sequence Ser-Phe-His-Val-Phe-Pro-Pro-Trp-Met-Cys-Lys-Xaa-Leu-Lys-Lys-Cys, (d). two peptides containing the consensus sequence Arg-Xaa-Cys-Trp-Lys-(Xaa)(2)-Asn-(Xaa)(3)-Val-Cys-Ser, (e). nine peptides containing the consensus sequence Ser-Leu-Pro-Ala-Gly-Leu-Ser-Pro, and (f). four peptides containing the consensus sequence Asp-Xaa-Gln-Asp-Arg-Trp-Xaa-Pro. The peptides did not inhibit the growth of Escherichia coli or Staphylococcus aureus and were inactive on hamster vascular or gastric smooth muscle preparations so that their biological functions, if any, remain to be established.