GC-NICI-MS analysis of acetazolamide and other sulfonamide (R-SO2-NH2)drugs as pentafluorobenzyl derivatives R-SO2-N(PFB)2 and quantification of pharmacological acetazolamide in human urine

摘要

Acetazolamide(molecular mass(MM),222)belongs to the class of sulfonamides(R-SO2-NH2)and is one of the strongest pharmacological inhibitors of carbonic anhydrase activity.Acetazolamide is excreted unchanged in the urine.Here,we report on the development,validation and biomedical application of a stable-isotope dilution GC-MS method for the reliable quantitative determination of acetazolamide in human urine.The method is based on evaporation to dryness of 50 mL urine aliquots,base-catalyzed derivatization of acetazolamide(d0-AZM)and its internal standard[acetylo-2H3]acetazolamide(d3-AZM)in 30 vol%pentafluorobenzyl(PFB)bromide in acetonitrile(60 min,30C),reconstitution in toluene(200 mL)and injection of 1-mL aliquots.The negative-ion chemical ionization(NICI)mass spectra(methane)of the PFB derivatives contained several intense ions including[M]‒at m/z 581 for d0-AZM and m/z 584 for d3-AZM,suggesting derivatization of their sulfonamide groups to form N,N-dipentafluorobenzyl derivatives(R-SO2-N(PFB)2),i.e.,d0-AZM-(PFB)2 and d3-AZM-(PFB)2,respectively.Quantification was performed by selected-ion monitoring of m/z 581 and 83 for d0-AZM-(PFB)2 and m/z 584 and 86 for d3-AZM-(PFB)2.The limits of detection and quantitation of the method were determined to be 300 fmol(67 pg)and 1 mM of acetazolamide,respectively.Intra-and inter-assay precision and accuracy for acetazolamide in human urine samples in pharmacologically relevant concentration ranges were determined to be 0.3%e4.2%and 95.3%e109%,respectively.The method was applied to measure urinary acetazolamide excretion after ingestion of a 250 mg acetazolamide-containing tablet(Acemit®)by a healthy volunteer.Among other tested sulfonamide drugs,methazolamide(MM,236)was also found to form a N,N-dipentafluorobenzyl derivative,whereas dorzolamide(MM,324)was hardly detectable.No GC-MS peaks were obtained from the PFB bromide derivatization of hydrochlorothiazide(MM,298),xipamide(MM,355),indapamide and metholazone(MM,366 each)or brinzolamide(MM,384).We demonstrate for the first time that sulfonamide drugs can be derivatized with PFB bromide and quantitated by GC-MS.Sulfonamides with MM larger than 236 are likely to be derivatized by PFB bromide but to lack thermal stability.