Green analytical method development for statin analysis.

Green analytical chemistry method was developed for pravastatin, fluvastatin and atorvastatin analysis. HPLC/DAD method using ethanol-based mobile phase with octadecyl-grafted silica with various grafting and related-column parameters such as particle sizes, core-shell and monolith was studied. Retention, efficiency and detector linearity were optimized. Even for column with particle size under 2 µm, the benefit of keeping efficiency within a large range of flow rate was not obtained with ethanol based mobile phase compared to acetonitrile one. Therefore the strategy to shorten analysis by increasing the flow rate induced decrease of efficiency with ethanol based mobile phase. An ODS-AQ YMC column, 50 mm × 4.6 mm, 3 µm was selected which showed the best compromise between analysis time, statin separation, and efficiency. HPLC conditions were at 1 mL/min, ethanol/formic acid (pH 2.5, 25 mM) (50:50, v/v) and thermostated at 40°C. To reduce solvent consumption for sample preparation, 0.5mg/mL concentration of each statin was found the highest which respected detector linearity. These conditions were validated for each statin for content determination in high concentrated hydro-alcoholic solutions. Solubility higher than 100mg/mL was found for pravastatin and fluvastatin, whereas for atorvastatin calcium salt the maximum concentration was 2mg/mL for hydro-alcoholic binary mixtures between 35% and 55% of ethanol in water. Using atorvastatin instead of its calcium salt, solubility was improved. Highly concentrated solution of statins offered potential fluid for per Buccal Per-Mucous(®) administration with the advantages of rapid and easy passage of drugs.

Development of a new sensitive and specific time-resolved fluoroimmunoassay (TR-FIA) of chlormadinone acetate in the serum of treated menopausal women.

We describe the development of a serum chlormadinone acetate (CMA) time-resolved fluoroimmunoassay (TR-FIA). We prepared haptens (3-CMO-chlormadinone acetate and 6-chloropregna-4,6-dien-17,20-diol-3-one-20-hemisuccinate), biotinylated tracers (3(biotinylaminopropylamido) 3-CMO-chlormadinone acetate and 3-(6-chloropregna-4,6-dien-17,20-diol-3-one-20-hemisuccinylamino)1-biotinylaminopropane), and immunogens necessary for eliciting two antibodies (anti-chlormadinone acetate 3-CMO/BSA and anti-chlormadinone 20-hemisuccinate/BSA). The specificity of the assay was rigorously studied to eliminate possible interference by polar metabolites of CMA, particularly 17 alpha-acetoxy-6-chloro-3beta-hydroxypregna-4,6-diene-20-one (3beta-hydroxy metabolite), employing an easy-to-use ethylene glycol chromatographic step prior to immunoassay, so as to separate the polar metabolites, in particular the 3beta-hydroxy-CMA metabolite, from the intact CMA. The choice of the anti-CMA antibody was guided by the high assay sensitivity obtained with the anti-CMA 3-CMO/BSA antibody. The detection limit was 51pg/ml. Interassay reproducibility CVs were between 2.6 and 4.5%. This TR-FIA thus appeared to be a sensitive, specific, precise, and consequently well-suited method for measurement of serum CMA during a pharmacokinetic study in women.

Comparison of oral versus transdermal testosterone supplementation in hypogonadal men.

OBJECTIVE: To compare mean serum total testosterone, bioavailable-testosterone, and dihydrotestosterone levels between transdermal testosterone and oral testosterone undecanoate treatment. METHODS: Multicentre, randomized, cross-over study; 44 men >18 years, testosterone ≤2.5 ng/mL. Two patches (Testopatch®) every other day in the morning or two capsules Pantestone® 40 mg bid in each 22-day period. Hormone serum levels of four blood samples over the first and last 48 h of each treatment period. RESULTS: Mean age 49 years. Mean testosterone before inclusion 1.99 ng/mL. Mean testosterone serum levels over the last 48 h of Testopatch treatment were superior to Pantestone (4.64 vs. 2.58 ng/mL, p<0.001). Testosterone trough levels at the end of each treatment period were significantly higher for Testopatch (3.15 vs. 2.45 ng/mL, p<0.01). Bioavailable-testosterone levels over the first and last 48 h of treatment were significantly greater with Testopatch than with Pantestone (p=0.001 and p<0.01). Dihydrotestosterone levels over the first and last 48 h of treatment (0.71 vs. 1.05 ng/mL and 0.68 vs. 0.89 ng/mL) as well as at trough (0.59 vs. 0.96 ng/mL) were significantly lower with Testopatch than with Pantestone (p<0.001, p<0.05, and p<0.001). SHBG levels decreased by Pantestone but not by Testopatch (p<0.001). CONCLUSIONS: Testopatch was superior to Pantestone to increase testosterone and bioavailable-testosterone levels in hypogonadal men from the first days and throughout the three weeks of treatment. Pantestone increased dihydrotestosterone to a larger extent and decreased SHBG.