, 3H, J = 7.1 Hz), 4.14.17 (m, 3H), 6.69.73 (m, 2H), 6.80 (d, 1H, J = 7.8 Hz), 7.16 (d, 1H, J = 7.7 Hz), 7.28 (t, 1H, J = 7.8 Hz), 7.34.74 (m, 9H), 7.86 (s, 1H), 12.5 (s, 1H); 13C NMR (CDCl3) 18.5, 32.9, 45.6, 115.4, 117.0, 118.1, 122.3, 123.4, 125.6, 126.6, 128.3, 128.3, 128.6, 129.2, 129.4, 129.7, 130.1, 131.9, 132.1, 132.5, 134.1, 135.6, 137.6, 138.0, 140.6, 143.1, 151.1, 172.9, 188.8, 196.5.Pharmaceutics 2013, 5 2.3. HPLC AnalysisA HPLC method was developed to simultaneously analyze the mono-naproxen co-drug 8, along with the parent compounds dithranol (1) and naproxen (5), and the dithranol derivatives 2 and 3. Samples were analyzed at ambient temperature using an Agilent 1100 series automated system with a quaternary solvent delivery system and a variable wavelength detector. The instrument was fitted with a Gemini C18, 5 m, 250 4.6 mm column (Phenomenex, Macclesfield, UK) and a Phenomenex Securityguard pre-column. The wavelength was set at 230 nm with a flow rate of 1 mL in-1 and the injection volume was 100 L. Two mobile phase compositions were used; mobile phase A was deionised water (adjusted with H3PO4 to pH 2.2) and mobile phase B was MeCN. A gradient mobile phase starting with H2O/H3PO4 (60:40) for 6.5 min, changing to H2O/H3PO4 (10:90) over 1 min, with the same condition running for 12.5 min and then returning to the initial conditions over 3.5 min. Calibration curves for each compound was constructed using the mobile phase and each provided R2 of 0.999. The retention times for 5, 2, 1, 3 and 8 were 6.5, 11.7, 12.4, 16.7 and 17.3 min respectively. The limits of detection (LoD) were 0.008, 0.45, 0.09, 1.8 and 0.9 g L-1 respectively. 2.4. Spectrophotometric Analysis Dithranol 1 and the co-drug 8 were diluted in 5 mL of MeCN to produce an equimolar (50 M) solution of each and measured quantitatively using a Hewlett Packard 8452A diode array spectrophotometer with 1 cm quartz cells, scanning from 190 to 1000 nm. The absorbance at 375 nm was recorded and all UV spectrophotometry experiments were carried out in triplicate. 2.5. Enzymatic Co-Drug Hydrolysis 2.5.1. Hydrolysis Using Porcine Liver Esterase Co-drug 8 was dissolved in acetonitrile at five concentrations, 91, 80, 69, 34 and 29 M and incubated with 120 IU mL-1 of porcine liver esterase (PLE) in phosphate buffered saline (PBS) and 5 acetonitrile to give a total reaction volume of 10 mL.Cinacalcet A magnetic stirrer was added and the reaction medium was constantly stirred.SMCC The solution was maintained at 25 .PMID:25147652 At regular intervals 400 L was withdrawn and 400 L of quenching solution (80 acetonitrile and 20 deionised H2O adjusted to pH 2.2 with H3PO4) was added. Samples were centrifuged at 12,000 rpm for 15 min, the supernatant was sampled and analyzed by HPLC. Control experiments contained 8 in an identical medium with the absence of PLE. The reactions were preformed in triplicate. 2.5.2. Hydrolysis Using Porcine Skin Homogenate Freshly excised porcine ears were immersed in Hanks buffer with ice during transport, before being washed with running tap water. Full thickness skin was isolated from underlying cartilage by blunt dissection using a scalpel. Hairs were removed with electric clippers. Skin samples (4 2 g) were cut into small pieces and placed in 15 mL PBS, before being homogenised using a high-shear laboratory mixer (Silverson Machines Ltd., UK) for 1 min. Co-drug 8 was first dissolved in an appropriate amount of acetonitrile to produce a final reaction solution with 80 M.