Analyzer Vitamin D3

Discussions about HPLC, CE, TLC, SFC, and other "liquid phase" separation techniques.

4 posts Page 1 of 1
Good afternoon,

I am facing difficulties in the analysis of vitamin D3 identification, in mineral matrix in the case (calcium carbonate), I am following the methodology of USP 40-NF35, follow the method.

Mobile phase: n-Hexane and isopropyl alcohol (99:1)
Standard solution: 2 µg/mL of USP Ergocalciferol RS or USP Cholecalciferol RS in n-hexane
System suitability solution: Heat a volume of Standard solution at 60° for 1 h to partially isomerize vitamin D (ergocalciferol or cholecalciferol) to its corresponding precursor.
Sample solution: Weigh NLT 20 Tablets, and grind the Tablets to a fine powder. Transfer the equivalent of 20 µg of cholecalciferol or ergocalciferol to a container having a polytef-lined screw cap. Add 8 mL of dimethyl sulfoxide and 12 mL of n-hexane, and shake for 45 min on a wrist-action shaker with tubes in a water bath maintained at 60°. Centrifuge for 10 min, withdraw the hexane layer by means of a pipet, and transfer to an evaporation flask. Add 12 mL of n-hexane to the dimethyl sulfoxide layer, mix on a vortex mixer for 5 min, and again withdraw the hexane layer by means of a pipet, and add to the evaporation flask. Repeat this extraction with three additional 12-mL portions of n-hexane, adding the hexane extracts to the evaporation flask. Evaporate the combined hexane extracts in vacuum at room temperature to dryness. Dissolve in and dilute the residue in a volume of n-hexane to obtain a concentration of 2 µg/mL.

Detector: UV 265 nm
Column: 4.6-mm × 15-cm; 5-µm packing L8
Flow rate: 1 mL/min
Injection size: 100 µL
What difficulties are you facing?
I find it difficult to extract the vit. D3, because the recovery is little, I have done several times the method, and I have a much lower recovery, already tested variation in the mobile phase, I already added in the citric acid extraction 100mg to see if the improvement has not yet succeeded ...

I would like to know if you have something, in vitamin D3 extraction, something concrete.
  using a zorbaxNh2 and DAD column for detection
It's hard for us to guess if the low recovery is due to the calibration, the sample preparation, integration, calculation,...

Extracting an apolar analyte (fat-soluble vitamin) from a polar matrix shouldn't give too much problems. The sample prep looks OK to me.

Why are you injecting such a large volume? How does the chromatography looks like?
4 posts Page 1 of 1

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