Preanalytical and analytical challenges in gas chromatographic determination of cholesterol synthesis and absorption markers

Tamara Gojkovic*, Sandra Vladimirov, Vesna Spasojevic-Kalimanovska, Aleksandra Zeljkovic, Jelena Vekic, Jelena Arsenijevic, Ivana Djuricic, Sladjana Sobajic, Zorana Jelic-Ivanovic

*Corresponding author for this work

Research output: Contribution to journalArticleResearchpeer-review

5 Citations (Scopus)


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.

Original languageEnglish
Pages (from-to)74-81
Number of pages8
JournalClinica Chimica Acta
Publication statusPublished - Mar 2018
Externally publishedYes


  • Freeze-thaw
  • Method validation
  • Plasticizers
  • Stability
  • Sterols


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