Development of method for determination of simvastatin and simvastatin acid in rat serum by liquid chromatography coupled with tandem mass spectrometry

Simvastatin je laktonsko predzdravilo, ki ima obsežen metabolizem prvega prehoda v jetrih. Njegov glavni presnovni produkt je simvastatinska kislina, ki je aktivna oblika simvastatina in deluje zaviralno na 3-hidroksi-3-metil glutaril koencim A reduktazo ter s tem zmanjša sintezo holesterola v jetrih. Simvastatin ima zaradi slabe vodotopnosti in obširnega metabolizma prvega prehoda zelo nizko biološko uporabnost. V podporo predklinični študiji na podganah, katere namen je primerjava farmakokinetičnih lastnosti novega predzdravila in različnih na UL-FFA razvitih formulacij s simvastatinom, smo želeli razviti občutljivo, selektivno, točno in natančno metodo za merjenje koncentracij simvastatina in simvastatinske kisline v majhnih (100 µL) volumnih podganjega seruma. Poskusili smo tri različne načine priprave vzorcev, tako da smo izvedli obarjanje proteinov, ekstrakcijo na trdnem nosilcu z različnimi kartušami in tekočinsko ekstrakcijo z različnimi organskimi topili. Vzorce smo analizirali s tekočinsko kromatografijo sklopljeno s tandemsko masno detekcijo, pri čemer smo primerjali tudi uporabo različnih kolon in njihov vpliv na odziv spojin. Razvoj metode je potekal na vzorcih s človeško plazmo, končni preizkus izbrane optimalne metode s tekočinsko ekstrakcijo s terc-butil metil etrom, pa smo izvedli na vzorcih s podganjim serumom. Selektivnost in natančnost metode smo ovrednotili na koncentracijskem območju med 0,5 µg/L in 50 µg/L. Ugotovili smo, da je simvastatin težavna spojina za analizo, ker ima problematično stabilnost in slabo ponovljivost priprave vzorcev. Boljšo ponovljivost je izkazovala simvastatinska kislina. Dokazali smo, da pri razviti metodi ni prisotnega učinka matrice, učinkovitost procesa priprave vzorca pa je okoli 15 % za simvastatin in 30 % za simvastatinsko kislino. Kljub nizkim vrednostim pa smo metodo preverili in zagotovili njeno ustreznost ter jo tudi potrdili v praksi na velikem številu vzorcev iz farmakokinetične študije.Simvastatin is a lactonic prodrug which has a large first pass metabolism in the liver. Its most important metabolite is simvastatin acid, an active form of simvastatin that is HMG-CoA reductase inhibitor and that causes decrease in cholesterol synthesis in liver. Because of the simvastatin’s low aqueous solubility and its large first pass metabolism simvastatin has extremely low bioavailability. The aim of our work was to develop a sensitive, accurate and precise method for determination of simvastatin and simvastatin acid in small aliquots of rat plasma (100 µL). The developed method should serve as analytical support the pharmacokinetic preclinical study, where a novel simvastatin prodrug and various simvastatin formulations were compared in terms of their pharmacokinetic properties To select the optimal sample preparation technique, three methods were tested: protein precipitation, solid phase extraction using different cartridges and liquid extraction using different organic solvents. The samples were analysed by liquid chromatography coupled to tandem mass spectrometry. Additionally two different columns and their influence on analyte response were tested and compared. The method development was performed using human plasma as a surrogate, while the final method which utilized the liquid-liquid extraction by terc-buthyl methyl ether was accomplished on rat serum. The selectivity, accuracy and precision were tested in concentration range between 0.5 µg/L and 50 µg/L. We concluded that simvastatin is a problematic analyte due to high variability during the sample preparation and poor stability. Better precision was achieved with simvastatin acid. We proved that the chosen method does not suffer from matrix effect, and the process efficiency is around 15% for simvastatin and around 30% for simvastatin acid. Regardless of the low process efficiency, the method has been successfully applied to a large number of study samples