| Abstract | Pirfenidon je novi antifibrotski, antioksidativni i protuupalni lijek registriran za terapiju idiopatske plućne fibroze. Uz to pokazuje značajan terapijski potencijal u srčanom, bubrežnom i jetrenom tkivu, zbog čega se i dalje istražuje. Za dio antifibrotskog učinka odgovoran je i njegov glavni metabolit, 5-karboksipirfenidon.
Cilj rada bio je razviti selektivnu i osjetljivu analitičku metodu za istovremenu analizu pirfenidona i 5-karboksipirfenidona unutar matriksa složenog biološkog uzorka, desnog ventrikla štakora tretiranog pirfenidonom.
U tekućinskoj kromatografiji visoke djelotvornosti korištena je stacionarna faza Symmetry (Waters) C18 (4,6 × 150 mm, 3,5 μm) pri temperaturi 28 °C. Ispitane su mobilne faze različitih omjera acetonitrila ili amonijevog formijata i vode. Isprobana je izokratna eluacija i gradijentno ispiranje, kao i različite brzine protoka. Najpovoljnijom se pokazala izokratna eluacija uz sastav ACN:H2O:HCOOH = 50:50:0,1% i brzinu protoka 0,4 mL/min, pri čemu su vremena zadržavanja pirfenidona i 5-karboksipirfenidona iznosila 5,9 i 4,3 min.
Za detekciju su korištena tri detektora: DAD, FLD i MS. DAD je poslužio u preliminarnoj optimizaciji HPLC metode, no za analizu složenih bioloških uzoraka nije bio dovoljno osjetljiv. FLD je pokazao potencijal primjene u detekciji oba spoja, ali zahtijeva daljnju optimizaciju. MS detektorom su analiti selektivno i osjetljivo detektirani u prisustvu kompleksnog matriksa, korištenjem kromatograma izoliranih iona m/z 186 za pirfenidon i m/z 216 za karboksipirfenidon, a također je proučena i njihova MS2 fragmentacija.
Paracetamol je odabran kao unutarnji standard na temelju povoljnog vremena zadržavanja (3,85 min) i svojstava (polarnosti i molekulske mase) bliskih analitima.
Za ekstrakciju analita isprobana su otapala čisti i 50%-tni acetonitril te čisti metanol. Također je dodatnom ekstrakcijom zaostalog taloga uzorka vodom provjerena učinkovitost navedenih otapala u izdvajanju polarnijeg 5-karboksipirfenidona. Utvrđeno je da je najbolje otapalo za pirfenidon metanol, dok je 50%-tni acetonitril također zadovoljavajuće ekstrakcijsko sredstvo. Za metabolit 5-karboksipirfenidon 50%-tni acetonitril je osigurao najveću ekstrakcijsku učinkovitost, a i metanol se pokazao prihvatljivim. Čisti acetonitril nije pogodno ekstrakcijsko sredstvo niti za jedan analit. |
| Abstract (english) | Pirfenidone is a novel antifibrotic, antioxidative and antiinflammatory agent registered for the therapy of idiopathic pulmonary fibrosis. In addition, it shows significant therapeutic potential in heart, kidney and liver tissue, due to which it is still under research. Its main metabolite, 5-carboxypirfenidone, is also partially responsible for the total antifibrotic action.
The aim of the work was to develop a selective and sensitive method for the analysis of pirfenidone and 5-carboxypirfenidone within the matrix of a complex biological sample, right rat ventricle.
In High Performance Liquid Chromatography, a Symmetry (Waters) C18 (4.6 × 150 mm, 3.5 μm) stationary phase was used, with the temperature of 28 °C. Mobile phases of various acetonitrile or ammonium formate vs. water ratios were tested, along with isocratic and gradient elution and different flow rates. Isocratic elution by a mobile phase consisting of ACN:H2O:HCOOH = 50:50:0.1% (V/V/V) at a 0.4 mL/min flow was chosen as best. The retention times of pirfenidone and 5-carboxypirfenidone under such conditions were 5.9 and 4.3 min, respectively.
For the detection, DAD, FLD and MS detectors were used. DAD was applied in the preliminary HPLC method optimisation, but proved insufficiently sensitive for the analysis of complex biological samples with low analyte concentrations. FLD showed a potential in detecting both compounds, although it requires further optimisation. MS detector enabled a selective and sensitive detection in the presence of a complex matrix by using Extracted Ion Chromatograms of m/z 186 for pirfenidone and 216 for 5-carboxypirfenidone. Also, their MS2 fragmentations were studied.
Paracetamol was chosen as the internal standard due to its favourable retention time (3.85 min) and the characteristics (polarity and molar mass) similar to the analytes.
As extraction solvents, pure and 50% acetonitrile along with pure methanol were tested. An additional extraction of the residual precipitate with water was performed in order to check the efficiency of the aforementioned solvents for the extraction of the more polar 5-carboxypirfenidone metabolite. Pure methanol turned out to be the most favourable solvent choice for pirfenidone, while 50% acetonitrile was also satisfactory. 50% acetonitrile provided highest extraction yields for 5-carboxypirfenidone, although methanol also proved acceptable. However, pure acetonitrile was not a good extraction solvent for either of the analytes. |
