· Salicylic Acid Is An Unpermitted Preservative.

Preparation Of Sample

· Non Alcoholic Beverages: – May Be Extracted Directly. If Troublesome Emulsion Forms During Extraction, Pipette 100 Ml Into A 250 Ml Volumetric Flask And Add 5 Gm Of Sod. Chloride And Shake Until Dissolved. Dilute To Volume With Alcohol, Shake Vigorously, Let Stand 10 Min, Shaking Occasionally, Filter And Take Filterate For

· Alcoholic Liquids: – Make 200 Ml Of Test Sample Alkaline To Litmus Paper With About 10 % Naoh Solution And Evaporate On Steam Bath To About ½ Its Original

· Solid Or Semisolid Substances: – Grind And Mix Thoroughly. Transfer 50 – 200 Gm According To The Consistency Of The Sample To 500 Ml Volumetric Flask, Add Water To Make About 400 Ml And Shake Until Mixture Becomes Uniform, Add 2- 5 Gm Calcium Chloride And Shake Until Dissolved. Make Distinctly Alkaline To Litmus Paper With About 10 % Na OH Solution, Dilute To Volume With Water, Shake Thoroughly, Let Stand 2 Hrs Shaking Frequently And

Qualitative Methods:

· Ferric Chloride Test: Salicylic Acid Is Extracted From The Acidified Food With Diethyl Ether And The Solvent Evaporated. The Residue Obtained On Evaporation Of The Ether Is Dissolved In Hot Water. On Treating This With 1 % Neutral Ferric Chloride Solution Salicylic Acid Gives Magenta

· Jorissen Test: Dissolve Residue From Ether Extract As Obtained In Method ‘A’ In Little Hot Cool 10 Ml Solution In Test Tube Add 4 Or 5 Drops Of 10% KNO2 Solution, 4 Or 5 Drops Of 50% Acetic Acid And One Drop Of 1% Cuso4 Solutions. Mix Thoroughly. Boil For A Few Minutes And Cool. Development Of Bordeaux Red Colour Indicates Presence Of Salicylic Acid. Benzoic Acid In Large Excess Gives A Buff Coloured Precipitate.

Estimation Of Salicylic Acid By HPLC-UV Method:

Principle

· Salicylic Acid Is Extracted From Known Quantity Of Foods Analysed By HPLC- UV Method

Reagents

· Anhydrous Ethanol AR Grade

· Methanol HPLC Grade

· Deionized Water (DI) ≥ 10 MΩ

· Sodium Chloride – AR Grade

· Phosphoric Acid – AR Grade

Preparation Of Sample

· Nonalcoholic Beverages: – May Be Extracted If Troublesome Emulsion Forms During Extraction, Pipette 100 Ml Into A 250 Ml Volumetric Flask And Add 5 Gm Of Sod. Chloride And Shake Until Dissolved. Dilute To Volume With Alcohol, Shake Vigorously, Let Stand 10 Min, Shaking Occasionally, Filter And Take Filterate For Test.

· Alcoholic Liquids: – Make 200 Ml Of Test Sample Alkaline To Litmus Paper With About 10 % Naoh Solution And Evaporate On Steam Bath To About ½ Its Original Dilute To Original Volume With Water And Filter If Necessary.

HPLC – UV Method:

· Column: Column – 15cm X 6mm ID, C-18, 5 Μm Particle Size

· Mobile Phase : Water/Methanol/Phosphoric Acid (60/40/0.2%)

· Elution: 0 Ml/Min Isocratic For 20 Min

· Detection: 254 Nm

Preparation Of Standard Solution:

· 400 Mg Of Salicylic Acid Is Dissolved In 50 Ml 70% Ethanol And Made Up To 100 Ml Volume With 70% Ethanol. Standard Dilutions Shall Be Made As And When Necessary From This Stock Solution.

· Injection – 10/20 µl Of Sample

The Samples Shall Be Injected In The Sequene

· Blank

· Standards

· Blank

· Reconstituted Extract From The Samples

· Quality Control Standards (20 µg/Ml And 60 µg/Ml Standards Samples Can Be Used For Quality Control)

Chromatogram:

· The Components Are Eluted In The Sequence, Benzoic Acid, Sorbic Acid, Methyl, Ethyl, Propyl And Butyl Parabens.

Calculations

· Calculate Concentration Of Each Preservative In Sample As Follows: Using Peak Areas Or Peak Heights And Concentrations Of Standards, Construct Linear Standard Curve For Each Compound Based On Formula Y = Mx + C, Where ‘X’ Is Concentration (Ppm), Y Is Peak Area Or Height, M Is Slope, And C Is The Intercept. Calculate Recovery Of Fortified Sample And Sample Results.

· Reference: D.P. Venema Et Al., J. Agric. Food Chem., 1996, 44, 1762-1767