A high pressure liquid chromatography procedure is reported for extraction and quantitation of 8 sulfonamides in stock solutions andin vitroplasma samples. This assay consists of a single, one-step extraction of sulfonamides from plasma and is sensitive to 10.0 ng/ml at 254 nm without additional concentration of the sample. Four sulfonamides (sulfamerazine, sulfamethazine, sulfapyridine and sulfathiazole) were separated from the plasma matrix by either mobile phase regardless of pH. The sulfonamides with the highest pKa, sulfanilamide (10.5) and sulfaguanidine (11.3), were only separable from plasma in a 50% water/50% methanol mobile phase at pH 7.45. The sulfonamide with the lowest pKa, sulfisoxazole (4.9), and its metabolite, acetylsulfisoxazole (N4), were separated from plasma by either mobile phase, 50/50 or 60/40 water/methanol, when acetate buffer reduced the pH to 4.00. Standard concentration curves of peak height were the most sensitive at 254 nm when a 60% water/40% methanol mobile phase at pH 4.00 was used. Sulfanilamide and sulfaguanidine were the most responsive to ultraviolet quantitation at 254 nm regardless of ionic suppression or polarity of the mobile phase.
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