ISO 12228-2:2014

Overview

ISO 12228-2:2014 defines a gas chromatographic method for the determination of individual and total sterols and triterpene dialcohols (erythrodiol, uvaol) specifically in olive oils and olive pomace oils. The standard covers sample preparation, saponification, unsaponifiable extraction, fraction separation, derivatization and quantitative gas chromatographic analysis to report sterol composition (%) and total sterol content (mg/kg).

Key topics and technical requirements

• Scope and applicability: Targets olive and olive pomace oils; other fats and oils are covered by ISO 12228‑1. The document is technically aligned with IOC Standard COI/T.20/Doc. No. 30 (2011).
• Sample preparation: Follow ISO 661 for test-sample preparation and ISO 5555 guidance for sampling; dry/filter samples if needed.
• Saponification and extraction: Boil the test portion with ethanolic KOH to saponify, then extract the unsaponifiable fraction with diethyl ether. (Safety warnings for flammability and hazardous reagents are included.)
• Fraction separation: Use thin‑layer chromatography (TLC) on basic silica gel plates to isolate the sterol and triterpene dialcohol fractions from the unsaponifiable matter.
• Derivatization: Convert sterols to trimethylsilyl (TMS) ethers using specified silylation reagents (e.g., HMDS/TMCS or BSTFA+TMCS) prior to GC.
• Gas chromatographic analysis: Use capillary column (20–30 m, 0.25–0.32 mm i.d.) coated with 5% diphenyl/95% dimethyl polysiloxane (SE‑52/SE‑54 or equivalent), split injection, flame ionization detector (FID), and cholestanol as the internal standard. Carrier gas: helium or hydrogen.
• Results expression: Report total sterol content in mg/kg and sterol composition as normalized percentage of peak areas. The standard specifies quantitative evaluation, composition reporting and precision criteria (repeatability/reproducibility) based on interlaboratory trials.
• Instrumentation and reagents: Lists required apparatus (rotary evaporator, TLC developing tanks, GC with data acquisition) and reagents (ethanolic KOH, diethyl ether, HMDS, TMCS, cholestanol).

Applications and users

• Who uses it: Food testing laboratories, olive oil producers, quality control teams, regulatory agencies, forensic and research labs focused on olive oil authenticity and compositional analysis.
• Practical uses:
  • Authenticity testing and detection of adulteration in olive oil.
  • Composition profiling for labeling, quality grading and certification.
  • Regulatory compliance and comparative studies of sterol profiles across olive cultivars or processing methods.

Related standards

• ISO 12228-1 - Determination of sterols in other animal and vegetable fats and oils.
• ISO 661 - Preparation of test sample (referenced normative).
• ISO 5555 - Sampling of olive oils (recommended guidance).
• IOC Standard COI/T.20/Doc. No. 30 - equivalent technical procedure.

Keywords: ISO 12228-2:2014, gas chromatographic method, sterols, triterpene dialcohols, olive oil, olive pomace oil, TLC, trimethylsilyl ethers, cholestanol, GC‑FID.