A Simple Method for Determination ofβ-Sitosterol as a Marker of Vegetable Oil Adulteration in Ghee by GC

A Simple Gas Chromatographic Method for Determination of β‑Sitosterol as a Marker of Vegetable Oil Adulteration in Ghee — Summary

Significance of the topic
Ghee (clarified butter) is a high‑value dairy fat widely used in food production and cooking. Because of its premium price, ghee is frequently adulterated with cheaper vegetable oils. β‑Sitosterol, a plant‑derived phytosterol abundant in vegetable oils but absent or at much lower levels in pure milk fat, is a robust chemical marker for detecting vegetable oil adulteration. A rapid, robust analytical method that reliably quantifies β‑sitosterol in ghee supports food safety, regulatory compliance, quality control and fraud detection in the dairy and food industries.

Objectives and overview of the study
The study aimed to develop and validate a simple, sensitive and cost‑effective gas chromatography (GC) method for extracting and quantifying β‑sitosterol in ghee, following the proposed FSSAI method No. 01.097:2022. The method was optimized to detect low levels of vegetable oil adulteration (down to 2% v/v) and to be appropriate for routine industry and research use. Extraction parameters and analytical conditions were investigated to maximize recovery and repeatability.

Methodology
Sample preparation:

• Accurately weigh 1 g of ghee.
• Saponify with 25 mL of 5% methanolic KOH and reflux at 95 °C for 45 minutes to release sterols.
• Cool and extract the reaction mixture three times with 15 mL portions of n‑hexane.
• Combine hexane extracts, dry over anhydrous sodium sulfate, wash with water, and evaporate solvent under a gentle nitrogen stream.
• Reconstitute residue in 0.5 mL n‑hexane, filter through 0.45 μm filter, and inject 0.5 μL into the GC.

Standard preparation and calibration:

• Prepare β‑sitosterol stock at 1000 mg/kg (10 mg in 10 mL chloroform), dilute with hexane to prepare seven calibration levels: 1, 2, 5, 10, 20, 50 and 100 mg/kg.

Extraction and method optimization:
The influence of extraction solvent type and volume, water volume, sonication time, surfactant type and concentration, and organic modifiers were evaluated to optimize extraction efficiency and sample cleanliness for GC analysis.

Used instrumentation
The analytical system and key GC conditions were:

• Shimadzu Nexis GC‑2030 equipped with flame ionization detector (FID).
• Autosampler: AOC‑20i/s (auto injector/auto sampler mentioned as related product).
• Capillary column: SH‑I‑Rxi‑5Sil MS, 30 m × 0.25 mm ID, df = 0.25 μm (P/N: 221‑75954‑30).
• Injection mode: Split, split ratio 15:1, injection volume 0.5 μL.
• Flow control: Constant flow (carrier gas nitrogen), column flow 0.7 mL/min.
• Injector temperature: 320 °C.
• Oven program: 140 °C (hold 2.0 min), ramp 15 °C/min to 320 °C, total ramp segment ~16 min; equilibration time 1.0 min.
• Detector: FID at 320 °C with gas flows Air 200 mL/min, H2 32 mL/min, N2 24 mL/min.

Main results and discussion

• Calibration and linearity: A seven‑point calibration from 1 to 100 mg/kg produced good linearity with regression coefficient R2 = 0.99.
• Sensitivity and detection limit: The method reliably detected vegetable oil adulteration in ghee down to 2% (v/v) adulteration; at this level β‑sitosterol produced S/N > 3, establishing practical detection capability for adulteration screening.
• Repeatability: At 10 mg/kg β‑sitosterol, retention time %RSD was 0.05% and peak area %RSD was 1.72% (n = 5), demonstrating good precision. More generally, replicate tests showed retention time RSD < 0.1% and area RSD < 5%.
• Recovery and accuracy: Spike recovery experiments in ghee at 20 and 50 mg/kg delivered recoveries of 91% and 98%, respectively; the broader recovery range observed in method testing was 70–120%, indicating acceptable matrix performance for routine screening when combined with suitable QC.
• Adulteration study: Controlled adulteration of ghee with vegetable oil at 2%, 5% and 10% yielded increasing β‑sitosterol responses consistent with expected trends, confirming applicability of the method to detect low‑level adulteration.

Benefits and practical applications

• The method uses straightforward saponification and hexane extraction followed by GC‑FID, avoiding expensive derivatization and high‑cost detectors.
• Sensitivity sufficient to detect 2% vegetable oil adulteration makes the approach suitable for routine QC, regulatory surveillance and in‑process monitoring.
• Good precision and acceptable recoveries support use in laboratories performing food authenticity testing and academic research on dairy fat composition.
• The method aligns with proposed FSSAI guidance, facilitating regulatory adoption in relevant jurisdictions.

Future trends and potential uses

• Extension to other sterol markers: Combining β‑sitosterol with a panel of plant sterols and triglyceride profiling could improve sensitivity and specificity for complex adulteration scenarios.
• Integration with mass spectrometry: GC‑MS or GC‑MS/MS could provide confirmatory identification and lower limits of detection for forensic investigations or when regulatory confirmation is required.
• High‑throughput automation: Automated sample preparation and autosamplers can increase throughput for large routine testing programs.
• Method harmonization and standardization: Wider inter‑laboratory validation and adoption into official compendia would strengthen regulatory enforcement against food fraud.

Conclusion
This work presents a simple, reproducible GC‑FID method on the Shimadzu Nexis GC‑2030 platform for quantifying β‑sitosterol in ghee. The procedure—including saponification, hexane extraction, and GC analysis—provides good linearity, precision and recovery, and detects vegetable oil adulteration at levels as low as 2% (S/N > 3). The approach is well suited for routine food industry analysis, regulatory screening and academic studies addressing ghee adulteration.

References

1. Kala A. et al., Determination of triacyl glycerol and sterol components of fat to authenticate milk fat based sweets. Journal of Food Science and Technology, 2016.
2. Food Safety and Standards Authority of India. Method for detection of adulteration in milk fat (clarified milk fat) with vegetable oils. File No. 1-90/FSSAI/SP (MS&A)/2009 dated 25 March 2018. Proposed method No. 01.097:2022.