TY - JOUR
T1 - Determination of non-cholesterol sterols in serum and HDL fraction by LC/MS-ms
T2 - Significance of matrix-related interferences Odrecrossed d signivanje neholesterolskih sterola u serumu i HDL frakciji upotrebom LC/MS-ms: Ispitivanje uticaja matriksa
AU - Vladimirov, Sandra
AU - Gojkovic, Tamara
AU - Zeljkovic, Aleksandra
AU - Jelic-Ivanovic, Zorana
AU - Spasojevic-Kalimanovska, Vesna
N1 - Funding Information:
Funding: This study was financially supported by a grant from the Ministry of Education, Science and Technological Development, Republic of Serbia (Project No. 175035).
Publisher Copyright:
© 2019 Walter de Gruyter GmbH, Berlin/Boston 2019.
PY - 2019/9/2
Y1 - 2019/9/2
N2 - 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 interferences and methods application in individual serum samples were examined. 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. 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 and 85.3-95.8% for HDL-NCS. 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.
AB - 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 interferences and methods application in individual serum samples were examined. 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. 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 and 85.3-95.8% for HDL-NCS. 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.
KW - HPLC-MS/MS
KW - calibration
KW - cholesterol precursors
KW - matrix effect
KW - phytosterols
UR - http://www.scopus.com/inward/record.url?scp=85074230855&partnerID=8YFLogxK
UR - https://pubmed.ncbi.nlm.nih.gov/33269018
U2 - 10.2478/jomb-2019-0044
DO - 10.2478/jomb-2019-0044
M3 - Article
AN - SCOPUS:85074230855
SN - 1452-8258
VL - 39
SP - 299
EP - 308
JO - Journal of Medical Biochemistry
JF - Journal of Medical Biochemistry
IS - 3
ER -