Testing Method For Vitamin D3

1. Appearance : Free flowing powder.

2. Color : Slightly yellowish or light yellow.

3. Odor : None to faint.

4. Dispersibility in water at 20^oC : Take 100 ml of distilled water cooled to 20^oC in a 250 mL Erlenmeyer flask, add 1 g of the sample and stir with a magnetic stirrer. If a homogeneous emulsion develops without solid particles after 5 mins, the sample corresponds.

5. Identity test for :

a.    Vitamin D3 : By HPLC.

b.    Tocopherol : By TLC.

Layer : Silica gel 60 F254, 0.25 mm.

Mobile phase : (Cyclohexane : Ether = 80 : 20) with chamber saturation.

Front distance : 15 cm.

Run time : 45 mins.

Sample solution : Transfer 500 mg of sample to a QF test tube add 4 ml of water and hold in ultrasonics for 2 min. Add 8 ml alcohol absolute and 20 ml n-hexane and shake for 30 secs, allow the phases to separate. Apply 5 µl from the upper phase.

Comparison solution :  Dissolve 20.0 mg of dl-alpha tocopherol in 100.0 ml of n-hexane and apply 10 µl. This solution is stable for one month when stored in refrigerator.

Detection : Dry the chromatogram in a cold air current for 2 mins.

                                (i)     Observe under UV light, decrease in in fluorescence at 254 nm or inherent

                                         fluorescence at 365 nm.

                                (ii)     Then spray with phosphomolybdic acid solution (1% in

                                         ethanol) and subsequently heat at 105^oC for 5 mins and estimate in

                                         daylight.

             The spots obtained by sample and comparison solution should have same Rf values.

Rf values & color :            Substance                          Rf                     (i)                  (ii)

of the spot                         Tocopherol                        0.35                   -                   blue

                                       

6.    Loss on drying : Dry approx. 1 g, accurately weighed, at 105^oC for 4 h. It loses not more than 8.0% of its weight.

7.    Assay, Vitamin D3 content :

Carry out the operations as rapidly as possible, avoiding exposure to actinic light and air.

Examine by HPLC.

Conditions :

                   Column : Purospher RP-18e @ 5 µm, 125 x 400 mm.

Wavelength : 280 nm.

Flow rate : 1.0 ml/min.

Retention time : 18 min.

Injected volume : 100 µl.

Mobile phase : (Methanol : H₂O = 90 : 3).

Standard solution  : Dissolve 10.0 mg of Vitamin D3 (potency : 100000 IU/g) with the mobile phase, stirrer for 25 mins and dilute to 100.0 ml with the mobile phase.

Sample solution : Dissolve 10.0 mg of the substance to be examined with the mobile phase, stirrer for 25 mins and dilute to 100.0 ml with the mobile phase.

Use the Mobile phase, Standard solution, and Sample solution (after filtering with 0.45 µm filter) for HPLC analysis.

Vitamin D3 100  contains not less than 100000 IU/g of Cholecalciferol.

    

8.    Powder fineness : Proceed with 40 g, manual/sieve shaker for 20 mins.

                                     Through sieve no. 22 : 100%.

                                     Through sieve no. 44 : Min. 90%.

                                     Through sieve no. 90 : Max. 15%.

9.    Microbial purity : Proceed with 25 g of sample for microbial purity (Ref : USP 30, according to

      specification of Corn Starch and Gum Acacia).

                                  A. Total aerobic microbial count : Max. 1000 cfu per g.

B.  Total combined molds and yeasts count : Max. 100 cfu per g.

C. Escherichia coli : Negative.

D. Staphylococcus aureus : Negative.

                                    E. Pseudomonas aeruginosa : Negative.

                                    F. Salmonella species : Negative.