Quantitation of mitomycin C in human ocular tissues by high-performance liquid chromatography-photo-diode array detection

A chromatographic method, which can quantitate mitomycin C (MMC) along with two antiglaucoma drugs, is described. The separation of MMC, alphagan and timolol was performed on a reversed-phase C,, column with water-methanol-tr ifluoroacetic acid (65:35:0.01, v/v) as the mobile phase. By monitoring at 360, 248 and 296 nm, the lower limits of detection for MMC, alphagan and ti molol are, respectively, 1.0, 2.0 and 5.0 ng (injection amount) at three-ti me S/N ratio. The dynamic ranges of quantitation for the three drugs are, r espectively, 1.0 ng-10.0 mug, 2.0 ng-10.0 mug and 5.0 ng-10.0 mug with line arity being larger than 0.9960. This method was applied to the determinatio n of MMC levels in Tenon's and trabeculum tissues of 10 glaucoma patients. MMC levels in these tissues, which were obtained from glaucoma filtering su rgery, were determined following a multiple extraction with methanol. The r ecovery of MMC for a two-batch extraction was better than 91.2%. The reprod ucibility of measurement for the MMC levels in these tissues is 2.5-6.0% RS D for triplicate injections. The intra-day variation of retention times for the MMC peaks was less than 1.6% RSD (n=3). The inter-day variation of ret ention times for the MMC peaks was less than 4.8% RSD (n=3). MMC was detect able in three trabeculum tissues out of 10 cases (ranging from 0.8 to 25.5 ng/mg specimen), while MMC was detected in nine Tenon's tissues out of 10 c ases (ranging from 0.3 to 21.1 ng/mg specimen). The results obtained show t hat the method is sensitive and selective for the quantitation of MMC. (C) 2001 Elsevier Science B.V. All rights reserved.