Razvoj in validacija metode za merjenje koncentracij rosuvastatina v plazemskih vzorcih bolnikov s prebolelim srčnim infarktom

Kemperle, Gašper (author), Trontelj, Jurij (mentor), Dermota, Tjaša (co-mentor)

Rosuvastatin predstavlja pomembno preventivo za ponovitev miokardnega infarkta pri bolnikih, ki so ga že preživeli. Rosuvastatin je kemijsko sintetiziran statin, ki selektivno, povratno ter kompetitivno zavira encim HMG-CoA reduktazo. Rosuvastatin je hidrofilen in ima od vseh statinov največjo afiniteto do aktivnega mesta encima ter štirikrat večjo od HMG-CoA. Študije so pokazale spremembo v farmokinetiki rosuvastatina pri hujših ledvičnih in jetrnih okvarah, prav tako v kombinaciji z drugimi zdravili. Spremembe v farmakokinetiki zaradi srčnega infarkta in sprememb v delovanju srčne mišice in možnih zapletov so dokazali tudi pri drugih zdravilih za preprečevanje srčno-žilnih boleznih (npr. atenolol in digoksin). V podporo farmakogenetski raziskavi v Univerzitetnem kliničnem centru Ljubljana (dovoljenje Komisije za medicinsko etiko RS št. 0120-124/2023/7) smo želeli razviti natančno, točno, občutljivo in selektivnometodo za merjenje koncentracij rosuvastatina v plazemskih vzorcih bolnikov. Poskusili smo tri različne načine priprave vzorcev: tekočinsko ekstrakcijo, podprto tekočinsko ekstrakcijo ter ekstrakcijo na trdnem nosilcu. Metodo, ki temelji na tekočinski ekstrakciji s terc-butil metil etrom in izopropilnim alkoholom v razmerju 9:1 ter detekcijo s tekočinsko kromatografijo, sklopljeno s tandemsko masno spektrometrijo (LC-MS/MS), smo validirali. Razvito metodo smo ovrednotili skladno s smernicami EMA in FDA. Točnost, ponovljivost in selektivnost smo potrdili v koncentracijskem območju med 0,3 ng/mL in 50 ng/mL. Izkoristek priprave vzorca je bil pri nizkem in visokem kontrolnemu vzorcu nad 60 %. Večji vpliv ozadja lahko pričakujemo pri zelo nizkih koncentracijah analita, vendar koncentracij, nižjih od 0,5 ng/mL, v realnih vzorcih ne pričakujemo. Morebitna prisotnost lipidnih delcev in krvi v plazmi ne bo vplivala na relativni vpliv ozadja. Ustreznost metode smo poleg uspešne validacije potrdili tudi na 84 vzorcih iz klinične raziskave. Vsi vzorci so bili znotraj določenega koncentracijskega območja med 1,1 in 37,3 ng/mL z relativno standardno deviacijo 78 %, povprečna plazemska koncentracija rosuvastatina je bila 12,06 ng/mL.

• Language: Slovenian
• Keywords: rosuvastatin, srčni infarkt, tekočinska ekstrakcija, LC-MS/MS
• Work type: Master's thesis/paper
• Organization: FFA - Faculty of Pharmacy
• Year: 2024
• PID: 20.500.12556/RUL-154475
• Publication date in RUL: 16.02.2024

Secondary language

• Language: English
• Title: Development and validation of a method for determination of rosuvastatin concentrations in plasma samples of post-myocardial infarction patients

Abstract

Rosuvastatin is an important treatment to prevent recurrent myocardial infarction (MI) in survivor patients. Rosuvastatin is a chemically synthesized statin that selectively, reversibly, and competitively inhibits the enzyme HMG-CoA reductase. Rosuvastatin is hydrophilic and has the highest affinity for the enzyme's active site among all statins, four times greater than that of HMG-CoA. The studies have shown changes in the pharmacokinetics of rosuvastatin in cases of severe kidney and liver impairments, as well as when used in combination with other medications. Changes in pharmacokinetics due to MI and alterations in the functioning of the myocardium, along with potential complications, have also been demonstrated with other medications for preventing cardiovascular diseases such as atenolol and digoxin. To support a pharmacogenetic study at the University Medical Centre Ljubljana (approval of National Medical Ethics Committee nr. 0120-124/2023/7), we aimed to develop a precise, accurate, sensitive, and selective method for measuring rosuvastatin concentrations in real plasma samples from MI patients. We attempted three different sample preparation methods: liquid-liquid extraction, supported liquid extraction, and solid-phase extraction. Method based on liquid-liquid extraction with tert-butyl methyl ether and isopropyl alcohol in a ratio of 9:1, with subsequent quantification by liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) was validated. We evaluated the developed method in accordance with the EMA and FDA guidelines. Accuracy, repeatability, and selectivity were confirmed in the concentration range between 0.3 ng/mL and 50 ng/mL. The sample preparation efficiency was over 60 % for both low and high-quality control samples. A more significant background impact can be expected at very low analyte concentrations, but such low concentrations (below 0.5 ng/mL) are not expected in patient samples. The potential presence of lipid particles and blood in plasma will also not affect the relative background impact. The suitability of the method was confirmed on 84 samples from a clinical study. All samples were within a specific concentration range between 1.1 and 37.3 ng/mL with a relative standard deviation of 78%. The average plasma concentration of rosuvastatin was 12.06 ng/mL.

• Keywords: rosuvastatin, heart attack, liquid-liquid extraction, LC-MS/MS