TY - JOUR
T1 - Preanalytical and analytical challenges in gas chromatographic determination of cholesterol synthesis and absorption markers
AU - Gojkovic, Tamara
AU - Vladimirov, Sandra
AU - Spasojevic-Kalimanovska, Vesna
AU - Zeljkovic, Aleksandra
AU - Vekic, Jelena
AU - Arsenijevic, Jelena
AU - Djuricic, Ivana
AU - Sobajic, Sladjana
AU - Jelic-Ivanovic, Zorana
N1 - Publisher Copyright:
© 2017
PY - 2018/3
Y1 - 2018/3
N2 - Introduction Cholesterol homeostasis disruption contributes to the development of different pathologies. Non-cholesterol sterols (NCSs) serve as cholesterol synthesis markers (desmosterol and lathosterol), and cholesterol absorption surrogate markers (campesterol, stigmasterol and β-sitosterol). The study aimed to resolve certain new pre-analytical and analytical problems and ensure a reliable and validated method. Materials and methods Method optimization, validation and stability studies were executed in human serum and plasma. Freeze-thaw cycles were done with and without antioxidant. Gas chromatography-mass spectrometer (GC-MS) was used for NCSs confirmation and plasticizer identification, while GC-flame ionization detector (GC-FID) was used for NCSs quantitation. Results Intra- and inter-assay variabilities for all NCSs were 2.75–9.55% and 5.80–7.75% for plasma and 3.10–5.72% and 3.05–10.92% for serum, respectively. Recovery studies showed satisfactory percentage errors for all NCSs: 93.4–105.7% in plasma and 87.5–106.9 in serum. Derivatized samples were stable up to 7 days at − 20 °C and derivatization yield was affected by presence of plasticizers. Fatty acid amids were identified as interfering plastic leachates. Statistically different NCSs concentrations were observed after the 1st freeze-thaw cycle, in antioxidant-free samples, and after the 4th cycle in antioxidant-enriched samples. Conclusions All of the in-house procedures proved to be useful for minimizing the preanalytical and analytical variations, as proven by the validation results.
AB - Introduction Cholesterol homeostasis disruption contributes to the development of different pathologies. Non-cholesterol sterols (NCSs) serve as cholesterol synthesis markers (desmosterol and lathosterol), and cholesterol absorption surrogate markers (campesterol, stigmasterol and β-sitosterol). The study aimed to resolve certain new pre-analytical and analytical problems and ensure a reliable and validated method. Materials and methods Method optimization, validation and stability studies were executed in human serum and plasma. Freeze-thaw cycles were done with and without antioxidant. Gas chromatography-mass spectrometer (GC-MS) was used for NCSs confirmation and plasticizer identification, while GC-flame ionization detector (GC-FID) was used for NCSs quantitation. Results Intra- and inter-assay variabilities for all NCSs were 2.75–9.55% and 5.80–7.75% for plasma and 3.10–5.72% and 3.05–10.92% for serum, respectively. Recovery studies showed satisfactory percentage errors for all NCSs: 93.4–105.7% in plasma and 87.5–106.9 in serum. Derivatized samples were stable up to 7 days at − 20 °C and derivatization yield was affected by presence of plasticizers. Fatty acid amids were identified as interfering plastic leachates. Statistically different NCSs concentrations were observed after the 1st freeze-thaw cycle, in antioxidant-free samples, and after the 4th cycle in antioxidant-enriched samples. Conclusions All of the in-house procedures proved to be useful for minimizing the preanalytical and analytical variations, as proven by the validation results.
KW - Freeze-thaw
KW - Method validation
KW - Plasticizers
KW - Stability
KW - Sterols
UR - http://www.scopus.com/inward/record.url?scp=85039173890&partnerID=8YFLogxK
U2 - 10.1016/j.cca.2017.12.032
DO - 10.1016/j.cca.2017.12.032
M3 - Article
C2 - 29274328
AN - SCOPUS:85039173890
SN - 0009-8981
VL - 478
SP - 74
EP - 81
JO - Clinica Chimica Acta
JF - Clinica Chimica Acta
ER -