Simultaneous quantification of carteolol and dorzolamide in rabbit aqueous humor and ciliary body by liquid chromatography/atmospheric pressure chemical ionization mass spectrometry

A rapid, sensitive and selective method for the simultaneous quantification of carteolol and dorzolamidein rabbit aqueous humor (AH) and ciliary body (CB) has been developed and validated usingreversed phase-high performance liquid chromatography (RP-HPLC) with isocratic elution coupled withatmospheric pressure chemical ionization mass spectrometry/mass spectrometry (APCI-MS/MS). Theanalytes and nadolol (used as internal standard, IS) were purified from AH by protein precipitation. Thesample preparation from CB was based on a two steps extraction procedure at different pH, utilizing aliquid–liquid extraction with a mixture of ethyl acetate, toluene and isopropanol 50:40:10 (v/v) at pH 8,followed by a second extraction with ethyl acetate at pH 11. The combined organic extracts were thenback extracted into 0.1% aqueous trifluoroacetic acid (TFA). The accuracy and precision values, calculatedfrom three different sets of quality control samples analyzed in sestuplicate on three different days, werewithin the generally accepted criteria for analytical methods (<15%). The assay proved to be accurate andprecise when applied to the in vivo study of carteolol and dorzolamide in rabbit AH and CB after singleadministration of an eye drops containing both drugs.