ANALYSIS OF CITRININ

ANALYSIS OF CITRININ

Reagents:
1. 4% KCL (4 gms KCL in 100 ml distilled water)
2. 20% H2SO4 (20 ml concentrated H2SO4 + 5% NaHC03( 5 gms of NaHC03 in 100 ml DW)
3. 6 N HCl (185.4 ml of HCl in 1 lt distilled water)
4. 10% Oxalic acid in methanol (W/V) (10 gm oxalic acid in 100 ml methanol)

Solvents: 
1. Acetonitrile
2. Iso octane
3. Chloroform
4. Chloroform – methanol – hexane (64:1:35)

Standards:
1. Citrinin : 10 g/ml in methanol

Procedure:

• Take 25 gms of sample, add 180 ml acetonitile, 20 ml 4% KCl, 2 ml 20% H2SO4 and shake for 15 minutes
• Filter and collect 100 ml of the filtrate
• Transfer filtrate to separating funnel and add 50ml iso octane. Shake for 1 minute.
• Collect the lower layer, add 50 ml iso octane and repeat the above step.
• Collect the lower layer into another separating funnel, add 25 ml water and extract with 50 ml chloroform
• Drain the chloroform layer into a separating funnel
• Extract again with two 10 ml portion of chloroform and combine all the three extracts
• Add 25 ml of 5% NaHC03 to the extract. Shake for 1 minute and drain off the lower portion.
• Re-extract with two 25 ml 5% NaHC03 portion and repeat the above step.
• Combine the extracted portion in a 600 ml beaker and acidify it with 6N HCl to pH 1-2, transfer it to separating funnel
• Rinse the beaker with 50 ml CHCl3, and transfer it to seperating funne, swirl for 30 seconds.
• Drain the lower portion, repeat above step with another 50 ml portion of CHCl3
• Collect the CHCl3 extracts and evaporate to near dryness
• Add 5 ml CHCl3 to the dried extract and filter through fluorocarbon filter into a 25 ml beaker
• Rinse the beaker twice with small amounts of CHCl3, filter and evaporate
• Dip the TLC plate in 10% oxalic acid solution for 2 min and air dry overnight
• Spot the sample and standard in 5-20 l range
• Develop plate in chloroform-methanol-hexane (64:1:35) for 45 minutes. Air dry and observe under long wave UV light for comparing the intensitites
• Calculate the citrinin content of sample using the following formula

Citrinin, ug/kg = S x Y x V X x W

Where,
           S = ul standard equal to unknown
           Y = Concentration of standard (ug/ml)
           V = Dilution of sample extract (ul)
           X = ul of sample spotted
           W= gms of sample represented by final extract

 = 25 x 100 = 12.376 g 202