Question

Which of the following tests ascertain detection of adulteration of ghee with vegetable oil by RP-HPLC method?

• A. Beta sitosterol test
• B. Baudouin test
• C. Hehner Test
• D. Holde Test

Hint:

To distinguish animal fats from plant fats, analyze their sterol profiles:
- Ghee (Animal) $\rightarrow$ Cholesterol.
- Vegetable Oil (Plant) $\rightarrow$ Phytosterols (principally \(\beta\)-sitosterol, campesterol, and stigmasterol).
RP-HPLC is the gold standard method for separating these sterol isomers.

Answer 1

The Correct Option is A

Step 1: Understanding the Concept:
Ghee (clarified butterfat) is a high-value dairy product susceptible to adulteration with cheaper vegetable fats and hydrogenated oils (vanaspati).
Vegetable fats contain unique plant sterols (phytosterols) that are absent in pure animal fats.
Detecting these marker compounds using modern chromatographic techniques is a standard method for identifying adulteration.

Step 2: Detailed Explanation:
Animal fats, including ghee, contain cholesterol as their primary sterol, with only trace or zero amounts of phytosterols.
In contrast, vegetable oils are rich in phytosterols, with \(\beta\)-sitosterol being the most abundant.
Pure ghee contains no \(\beta\)-sitosterol.
Therefore, the presence of \(\beta\)-sitosterol in a ghee sample is a definitive indicator of adulteration with vegetable oils or fats.
To detect this, the unsaponifiable matter of the ghee sample is extracted and analyzed using Reversed-Phase High-Performance Liquid Chromatography (RP-HPLC) or Gas Chromatography (GC).
The RP-HPLC method separates the sterols based on their hydrophobic interactions with the stationary phase.
This allows for the precise separation and quantification of \(\beta\)-sitosterol from cholesterol.
Even low levels of vegetable oil contamination (down to $1\%$) can be detected by identifying the \(\beta\)-sitosterol peak in the chromatogram.
Let us review the other options:
- The Baudouin test is a colorimetric test used specifically to detect sesame oil (using furfural and hydrochloric acid), but it is not an HPLC-based method.
- Hehner's test is used to detect formaldehyde in milk.
- Holde's test is a physical-chemical test used to detect mineral oil contamination in vegetable oils.

Step 3: Final Answer:
The \(\beta\)-sitosterol test by RP-HPLC is used to detect the adulteration of ghee with vegetable oil.