Validirane RP-RP-HPLC i TLC metode za simultano određivanje tamsulozin hidroklorida i finasterida u istom dozirnom pripravku

Reversed phase high-performance liquid chromatography (RP-HPLC) and thin-layer chromatography (TLC) methods have been developed and validated for simultaneous estimation of tamsulosin hydrochloride and finasteride in bulk drug and in combined dosage forms. RP-HPLC separation was achieved on a Phenomenex C18 column using methanol/0.02 mol L-1 ammonium acetate buffer/triethylamine (79.9 + 20 + 0.1, V/V/V) (pH 9.2) as mobile phase. The TLC separation was achieved on an aluminium-backed layer of silica gel 60F254 using toluene/methanol/triethylamine (9 + 1.5 + 1, V/V/V) as eluent. Quantification was achieved with photodiode array (PDA) detection at 235 nm over the concentration range 0.5–16 and 150 µg mL1 with mean recovery of 99.8 ± 0.9 and 100.0 ± 0.8 % for tamsulosin hydrochloride and finasteride, respectively, by the RP-HPLC method. Quantification was achieved with UV detection at 270 nm over the concentration range 100–2000 ng per spot and 250–5000 ng per spot with mean recovery of 98.9 ± 0.9 and 99.6 ± 0.7 % for tamsulosin hydrochloride and finasteride, respectively, by the TLC method. Both methods are simple, precise, accurate and sensitive and are applicable to the simultaneous determination of tamsulosin hydrochloride and finasteride in bulk drug and in combined dosage forms.U radu su opisani razvoj i validacija inverzno fazne kromatografije visoke učinkovitosti (RP-HPLC) i tankoslojne kromatografije (TLC) za simultano određivanje tamsulozin hidroklorida i finasterida kao čistih supstancija i u kombiniranim tabletama. Za RP-HPLC odjeljivanje korištena je Phenomenex C18 kolona (250 mm, 4,6 mm, 5 µm) i metanol/0,02 mol L–1 pufer s amonijevim acetatom/trietilamin (79,9+20+0,1, V/V/V) (pH 9,2) kao pokretna faza, pri protoku 1 mL min-1. TLC odjeljivanje rađeno je na silikagelu 60F254 na aluminijskoj podlozi, koristeći toluen/metanol/trietilamin (9+1,5+1, V/V/V) kao eluens. Za detekciju u RP-HPLC metodi korištena je fotodioda (PDA) pri 235 nm te je provedena kvantitacija u koncentracijskom području 0,5–16 µg mL–1 i 1–50 µg mL–1, uz srednji analitički povrat od 99,8 ± 0,9 % za tamsulozin hidroklorid i 100,0 ± 0,8 % za finasterid. Za kvantitaciju u TLC metodi korištena je UV detekcija pri 270 nm u koncentracijskom području 100–2000 ng po točki za tamsulozin hidroklorid i 250–5000 ng po točki za finasterid, uz srednji analitički povrat od 98,9 ± 0,9, odnosno 99,6 ± 0,7 %. Obje metode su jednostavne, precizne, točne i osjetljive i mogu se primijeniti za simultano određivanje tamsulozin hidroklorida i finasterida kao čistih supstancija i u kombiniranim dozirnim oblicima

Simultaneous pharmacokinetic and pharmacodynamic analysis of 5α-reductase inhibitors and androgens by liquid chromatography tandem mass spectrometry

AbstractBenign prostatic hyperplasia and prostate cancer can be treated with the 5α-reductase inhibitors, finasteride and dutasteride, when pharmacodynamic biomarkers are useful in assessing response. A novel method was developed to measure the substrates and products of 5α-reductases (testosterone, 5α-dihydrotestosterone (DHT), androstenedione) and finasteride and dutasteride simultaneously by liquid chromatography tandem mass spectrometry, using an ABSciex QTRAP® 5500, with a Waters Acquity™ UPLC. Analytes were extracted from serum (500µL) via solid-phase extraction (Oasis® HLB), with 13C3-labelled androgens and d9-finasteride included as internal standards. Analytes were separated on a Kinetex C18 column (150×3mm, 2.6µm), using a gradient run of 19min. Temporal resolution of analytes from naturally occurring isomers and mass +2 isotopomers was ensured. Protonated molecular ions were detected in atmospheric pressure chemical ionisation mode and source conditions optimised for DHT, the least abundant analyte. Multiple reaction monitoring was performed as follows: testosterone (m/z 289→97), DHT (m/z 291→255), androstenedione (m/z 287→97), dutasteride (m/z 529→461), finasteride (m/z 373→317). Validation parameters (intra- and inter-assay precision and accuracy, linearity, limits of quantitation) were within acceptable ranges and biological extracts were stable for 28 days. Finally the method was employed in men treated with finasteride or dutasteride; levels of DHT were lowered by both drugs and furthermore the substrate concentrations increased