Determination of non-cholesterol sterols in serum and HDL fraction by LC/MS-ms: Significance of matrix-related interferences

Abstract

Background:Non-cholesterol sterols (NCS) are promising biomarkers for estimation of cholesterol homeostasis properties. In addition, determination of NCS in high-density lipoprotein (HDL) fraction (HDL-NCS) could provide information on cholesterol efflux. However, matrix effects interfere in liquid chromatography–mass spectrometry (LC-MS)analysis of NCS, thereby impairing the method sensitivity.The aims of this study were development, optimization and validation of LC-MS method for quantification of NCS in serum and HDL-NCS. Additionally, matrix effect interfer-ences and methods application in individual serum samples were examined.Methods:HDL precipitating reagent was used for HDL isolation. Matrix effect was examined by comparing different surrogates by simple regression analysis. Validation was conducted according to the FDA-ICH guideline. 20 healthy volunteers were recruited for testing of method application.Results:The observed matrix effect was 30%, and matrix comparison showed that cholesterol was the dominant contributor to the matrix effect. Cholesterol concentration was adjusted by construction of the calibration curve for serum and HDL fraction (5 mmol/L and 2.5 mmol/L,respectively). The intra- and inter- run variabilities for NCSs were 4.7–10.3% for serum NCS and 3.6–13.6% for HDL-NCS and 4.6–9.5% for serum NCSs and 2.5–9.8% for HDL-NCS, respectively. Recovery studies showed satisfactory results for NCSs: 89.8–113.1% for serum NCS and85.3–95.8% for HDL-NCS. Conclusions:The method was successfully developed and optimized. The matrix interference was solved by customising calibration curves for each method and sample type.The measurement of NCS in HDL fraction was proposed for the first time as potentially useful procedure in biomedical researches.

Uvod: Neholesterolski steroli (NHS) predstavljaju potencijalne biomarkere homeostaze holesterola. Pored toga, određivanje NHS u HDL frakciji (HDL-NHS) moglo bi da pruži dodatne informacije o efluksu holesterola. Međutim, određivanje NHS metodom tečne hromatografije - masene spektrometrije (LC-MS) je podložno uticaju matriksa usled čega dolazi do sniženja osetljivost metode. Studija je prvenstveno imala za cilj razvoj, optimizaciju i validaciju LC-MS metode za kvantifikaciju NHS u serumu i HDL-NHS. Pored toga, detaljno je ispitan efekat matriksa i metoda primenjena za analizu uzoraka seruma zdravih dobrovoljaca. Metode: Izolacija HDL frakcije je izvršena upotrebom precipitirajućeg reagensa. Efekat matriksa je ispitan poređenjem različitih surogat matriksa regresionom analizom. Validacija je sprovedena u skladu sa FDA-ICH vodičem za validaciju bioanalitičkih metoda. Korišćeni su serumi 20 zdravih dobrovoljaca u cilju kliničke evaluacije metode. Rezultati: Efekat matriksa je iznosio 30% i dalja analiza je pokazala da je holesterol bio glavni uzročnik efekta matriksa. U cilju rešavanja ove interferencije, kalibracione krive za su pripremljene u metanolnom rastvoru holesterola koncentracije 5 mmol/L, a za kvantifikaciju HDL-NHS u metanolnom rastvoru holesterola koncentracije 2,5 mmol/L. Varijacije unutar serije su bile 4,7-10,3% za NHS i 3,6-13,6% za HDL-NHS, a između serija 4,6-9,5% za NHS i 2,5-9,8% za HDL-NHS. Studije prinosa analita su pokazale zadovoljavajuće rezultate: 89,8-113,1% za NHS i 85,3-95,8% za HDL-NHS. Zaključak: Metoda je uspešno optimizovana i validirana. Interferencija usled prisustva holesterola je korigovana upotrebom odgovarajućih kalibracionih krivih za svaku vrstu uzorka. Određivanje HDL-NHS je po prvi put predloženo kao potencijalno korisna procedura u biomedicinskim istraživanjima.