Estimation of β-Sitosterol in Milk Fat (Ghee) Samples

Significance of the topic

Ghee, or clarified butter, is a staple in culinary and traditional applications, but widespread adulteration with vegetable oils undermines product quality and poses regulatory concerns. Plant sterols, notably β-sitosterol, represent reliable markers for detecting non-dairy lipids in milk fat. Effective monitoring of β-sitosterol enables food safety laboratories to ensure authenticity and comply with quality standards.

Aims and overview of the study

This work presents the development and validation of a gas chromatography–mass spectrometry method using an Agilent 8890 GC coupled to a 5977B single quadrupole MS for the precise detection and quantification of β-sitosterol in ghee. The approach targets routine testing in industrial, commercial, and academic settings.

Methodology and instrumentation

Sample preparation entailed:

• Saponification of 200 mg fat with 10 M KOH and 20% ascorbic acid
• Liquid–liquid extraction of unsaponifiable matter using n-hexane and water
• Derivatization of extracted sterols to trimethylsilyl ethers with BSTFA in pyridine at 80 °C for 40 min

Chromatographic separation was performed on a non-polar capillary column (30 m × 0.25 mm × 0.25 μm) with a temperature program from 80 °C (1 min) to 290 °C (30 min) at 15 °C/min. MS detection employed electron ionization in selected ion monitoring mode, tracking ions at m/z 396, 486, 357, 381, and 129.

Main results and discussion

The method achieved a limit of quantitation (LOQ) of 200 ppb, with signal-to-noise ratios above 35 at this level and no interfering signals in blanks. Calibration over 200 ppb–5 ppm exhibited excellent linearity (R2 > 0.999). Six replicate injections at 1 ppm yielded an RSD of 2.48%, demonstrating high repeatability. Analysis of a commercial ghee sample detected 2.24 ppm β-sitosterol. Recovery studies at spiking levels of 5 ppm and 25 ppm provided recoveries of 125.6% and 83.4%, respectively, confirming method accuracy.

Benefits and practical applications

This analytical workflow offers:

• High confidence in routine authenticity testing of milk fat
• Relatively simple sample preparation with common laboratory reagents
• Rapid analysis suitable for quality control laboratories
• Compatibility with standard GC/MS instrumentation

Future trends and applications

Emerging opportunities include automating sample handling for high-throughput screening, coupling with high-resolution mass spectrometry for broader sterol profiling, extending the approach to other lipid biomarkers, and deploying portable GC/MS platforms for on-site authenticity testing.

Conclusion

A robust GC/MS-SIM method has been established for precise quantitation of β-sitosterol in ghee, enabling detection of vegetable oil adulteration with high sensitivity, linearity, and reproducibility. The protocol supports comprehensive quality assurance in food analysis.

Instrumentation used

• Agilent 8890 GC system with split/splitless inlet
• Agilent 5977B single quadrupole mass spectrometer
• Agilent J&W HP-5ms Ultra Inert capillary column (30 m × 0.25 mm, 0.25 μm)
• BSTFA derivatization reagent, pyridine, n-hexane, KOH, ascorbic acid

References

1. Rani A et al. A rapid reversed-phase thin layer chromatographic protocol for detection of adulteration in ghee with vegetable oils. Journal of Food Science and Technology. 2015;52(4):2434–2439.
2. Jyotika. Profiling of milk fat of different species of milch animals. NDRI, Karnal; 2016. P-1452.
3. Food Safety and Standards Authority of India. Methods of detecting adulteration in ghee. Food Safety Mantra. 2019;May 27.