High-performance liquid chromatographic assay for pilocarpine in aqueous humor.
A high-performance liquid chromatographic assay for pilocarpine has been developed for the determination of pilocarpine in aqueous humor. A structurally similar internal standard is used, and pilocarpine is separated from isopilocarpine under the chromatographic conditions used. A 100-microliter sample is mixed with an aliquot of internal standard at pH 8.3 and extracted with methylene chloride. The extract is evaporated to dryness and the alkaloids are quaternized with p-nitrobenzyl bromide. Following the quaternization, the sample is evaporated to dryness, washed and diluted with a mobile phase--triethylamine mixture and analyzed by high-performance liquid chromatography using a reversed-phase octadecylsilane column with detection at a wavelength of 254 nm. This is a highly sensitive, reproducible and selective assay for measuring pilocarpine at physiological levels in individual aqueous humor samples.
D000818:Animals; D001082:Aqueous Humor / Q000032:analysis*; D002851:Chromatography, High Pressure Liquid / Q000379:methods; D007536:Isomerism; D008297:Male; D010862:Pilocarpine / Q000031:analogs & derivatives / Q000032:analysis*; D011817:Rabbits
D010862:Pilocarpine; C029118:pilocarpic acid