ABSTRACT
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 μL 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, 30 °C), reconstitution in toluene (200μL) and injection of 1-μL 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-dipenta-fluorobenzyl derivatives (R-SO2-N(PFB)2), i.e., d0-AZM-(PFB)2 and d3-AZM-(PFB)2, respectively. Quanti-fication 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 1μM 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％-4.2％and 95.3％-109％, 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 quan-titated by GC-MS. Sulfonamides with MM larger than 236 are likely to be derivatized by PFB bromide but to lack thermal stability.
ABSTRACT
S-Nitrosothiols or thionitrites with the general formula RSNO are formally composed of the nitrosylcation(NO+)and a thiolate(RS-),the base of the corresponding acids RSH.The smallest S-nitrosothiol isHSNO and derives from hydrogen sulfide(HSH,H2S).The most common physiological S-nitrosothiols arederived from the amino acid L-cysteine(CysSH).Thus,the simplest S-nitrosothiol is S-nitroso-L-cysteine(CysSNO).CysSNO is a spontaneous potent donor of nitric oxide(NO)which activates soluble guanylylcyclase to form cyclic guanosine monophosphate(cGMP).This activation is associated with multiplebiological actions that include relaxation of smooth muscle cells and inhibition of platelet aggregation.Like NO,CysSNO is a short-lived species and occurs physiologically at concentrations around 1 nM inhuman blood.CysSNO can be formed from CysSH and higher oxides of NO including nitrous acid(HONO)and its anhydride(N2O3).The most characteristic feature of RSNO is the S-transnitrosation reaction bywhich the NO+group is reversibly transferred to another thiolate.By this way numerous RSNO can beformed such as the low-molecular-mass S-nitroso-N-acetyl-L-cysteine(SNAC)and S-nitroso-glutathione(GSNO),and the high-molecular-mass S-nitrosol-L-cysteine hemoglobin(HbCysSNO)present in erythrocytesand S-nitrosol-L-cysteine albumin(AlbCysSNO)present in plasma at concentrations of theorder of 200 nM.All above mentioned RSNO exert NO-related biological activity,but they must be administeredintravenously.This important drawback can be overcome by lipophilic charge-free RSNO.Thus,we prepared the ethyl ester of SNAC,the S-nitroso-N-acetyl-L-cysteine ethyl ester(SNACET),fromsynthetic N-acetyl-L-cysteine ethyl ester(NACET).Both NACET and SNACET have improved pharmacologicalfeatures over N-acetyl-L-cysteine(NAC)and S-nitroso-N-acetyl-L-cysteine(SNAC),respectively,including higher oral bioavailability.SNACET exerts NO-related activities which can be utilized in theurogenital tract and in the cardiovascular system.NACET,with high oral bioavailability,is a strong antioxidantand abundant precursor of GSH,unlike its free acid N-acetyl-L-cysteine(NAC).Here,we reviewthe chemical and pharmacological properties of SNACET and NACET as well as their analytical chemistry.We also report new results from the ingestion of S-[15N]nitroso-N-acetyl-L-cysteine ethyl ester(S15NACET)demonstrating the favorable pharmacological profile of SNACET.