Assay—
[NOTE—Throughout this
Assay, protect solutions containing, and derived from, the test specimen and the Reference Standard from the atmosphere and light, preferably by the use of a blanket of inert gas and low-actinic glassware.
]
Ether—
Use ethyl ether. Use within 24 hours after opening container.
Dehydrated hexane—
Prepare a chromatographic column by packing a chromatographic tube, 60 cm × 8 cm in diameter, with 500 g of 50- to 250-µm chromatographic siliceous earth, activated by drying at 150
for 4 hours (see
Column adsorption chromatography under
Chromatography 
621
). Pass 500 mL of hexanes through the column, and collect the eluate in a glass-stoppered flask.
Butylated hydroxytoluene solution—
Dissolve a quantity of butylated hydroxytoluene in chromatographic hexane to obtain a solution containing 10 mg per mL.
Aqueous potassium hydroxide solution—
Dissolve 500 g of potassium hydroxide in 500 mL of freshly boiled water, mix, and cool. Prepare this solution fresh daily.
Alcoholic potassium hydroxide solution—
Dissolve 3 g of potassium hydroxide in 50 mL of freshly boiled water, add 10 mL of alcohol, dilute with freshly boiled water to 100 mL, and mix. Prepare this solution fresh daily.
Sodium ascorbate solution—
Dissolve 3.5 g of ascorbic acid in 20 mL of 1 N sodium hydroxide. Prepare this solution fresh daily.
Sodium sulfide solution—
Dissolve 12 g of sodium sulfide in 20 mL of water, dilute with glycerin to 100 mL, and mix.
Mobile phase—
Prepare a 3 in 1000 mixture of n-amyl alcohol in Dehydrated hexane. The ratio of components and the flow rate may be varied to meet system suitability requirements.
Standard preparation—
Transfer about 25 mg of
USP Ergocalciferol RS, accurately weighed, to a 50-mL volumetric flask, dissolve without heat in toluene, add toluene to volume, and mix. Prepare stock solution fresh daily.
Assay preparation—
Reflux not less than 10 Capsules with a mixture of 10 mL of
Sodium ascorbate solution and 2 drops of
Sodium sulfide solution on a steam bath for 10 minutes, crush any remaining solids with a blunt glass rod, and continue heating for 5 minutes. Cool, add 25 mL of alcohol and 3 mL of
Aqueous potassium hydroxide solution, and mix.
Reflux the mixture on a steam bath for 30 minutes. Cool rapidly under running water, and transfer the saponified mixture to a conical separator, rinsing the saponification flask with two 15-mL portions of water, 10 mL of alcohol, and two 50-mL portions of ether. Shake the combined saponified mixture and rinsings vigorously for 30 seconds, and allow to stand until both layers are clear. Transfer the aqueous phase to a second conical separator, add a mixture of 10 mL of alcohol and 50 mL of solvent hexane, and shake vigorously. Allow to separate, transfer the aqueous phase to a third conical separator, and transfer the hexane phase to the first separator, rinsing the second separator with two 10-mL portions of solvent hexane, adding the rinsings to the first separator. Shake the aqueous phase in the third separator with 50 mL of solvent hexane, and add the hexane phase to the first separator. Wash the combined ether-hexane extracts by shaking vigorously with three 50-mL portions of
Alcoholic potassium hydroxide solution, and wash with 50-mL portions of water vigorously until the last washing is neutral to phenolphthalein. Drain any remaining drops of water from the combined ether-hexane extracts, add 2 sheets of 9-cm filter paper, in strips, to the separator, and shake. Transfer the washed ether-hexane extracts to a round-bottom flask, rinsing the separator and paper with solvent hexane. Combine the hexane rinsings with the ether-hexane extracts, add 100 µL of
Butylated hydroxytoluene solution, and mix. Evaporate in vacuum to dryness by swirling in a water bath maintained at a temperature not higher than 40
. Cool under running water, and introduce nitrogen sufficient to restore atmospheric pressure. Without delay, dissolve the residue in a measured volume of a 1 in 5 mixture of toluene and
Mobile phase, until the concentration of vitamin D is about 25 µg per mL, to obtain the
Assay preparation.
Chromatographic system—
Use a chromatograph, operated at room temperature, fitted with an UV detector that monitors absorption at 254 nm, and a 25-cm × 4.6-mm stainless steel column packed with column packing L3.
System suitability test—
Transfer about 100 mg of
USP Vitamin D Assay System Suitability RS to a 10-mL volumetric flask, add a 1 in 5 mixture of toluene and
Mobile phase to volume, and mix. Heat a portion of this solution under reflux, at 90
for 45 minutes, and cool. Chromatograph five injections of this solution, and measure the peak responses as directed under
Procedure: the relative standard deviation for the cholecalciferol peak response does not exceed 2.0%, and the resolution between
trans-cholecalciferol and pre-cholecalciferol is not less than 1.0.
[NOTE—Chromatograms obtained as directed for this test exhibit relative retention times of approximately 0.4 for pre-cholecalciferol, 0.5 for
trans-cholecalciferol, and 1.0 for cholecalciferol.
]
Vitamin D response factor—
Transfer 4.0 mL of the
Standard preparation to a 100-mL volumetric flask, dilute with
Mobile phase to volume, and mix to obtain the
Working standard preparation. Store this
Working standard preparation at a temperature not above 0
, retaining the unused portion for the
Procedure. Inject 20 µL of the
Working standard preparation into the column, and measure the peak response for vitamin D. Calculate the response factor,
FD, by the formula:
CS / rS,
in which
CS is the concentration, in µg per mL, of ergocalciferol in the
Working standard preparation, and
rS is the peak response of ergocalciferol.
Pre-vitamin D response factor—
Pipet 4 mL of the
Standard preparation into a round-bottom flask fitted with a reflux condenser, and add 2 or 3 crystals of butylated hydroxytoluene. Displace the air with nitrogen, and heat in a water bath maintained at a temperature of 90
in subdued light under a nitrogen atmosphere for 45 minutes, to obtain a solution containing vitamin D and pre-vitamin D. Cool, transfer with the aid of several portions of
Mobile phase to a 100-mL volumetric flask, dilute with
Mobile phase to volume, and mix to obtain the
Working mixture. Inject 20 µL of this
Working mixture into the analytical column, and measure the peak responses for vitamin D and pre-vitamin D. Calculate the concentration,
C¢S, in µg per mL, of vitamin D in the (heated)
Working mixture taken by the formula:
FDr¢S,
in which
r¢S is the peak response for vitamin D. Calculate the concentration,
C¢pre, in µg per mL, of pre-vitamin D, in the
Working mixture taken by the formula:
C¢pre =
CS 
C¢S.
Calculate the response factor,
Fpre, for pre-vitamin D by the formula
C¢pre/r¢pre, in which
r¢pre is the peak response of pre-vitamin D.
[NOTE—The value of
Fpre determined in duplicate, on different days, can be used during the entire procedure.
]
Procedure—
Inject 20 µL of the
Assay preparation into the column, and measure the peak responses for vitamin D and pre-vitamin D. Calculate the concentration, in µg per mL, of C
28H
44O in the
Assay preparation taken by the formula:
r¢¢DFD+ r¢¢preFpre,
in which
r¢¢D and
r¢¢pre are the peak responses of vitamin D and pre-vitamin D, respectively.
