Identification—
A:
Transfer a portion of finely powdered Tablets,equivalent to about 10mg of phytonadione,to a 1000-mLvolumetric flask,add 750mLof dehydrated alcohol,and shake vigorously.Dilute with dehydrated alcohol to volume,mix,and filter:the UVabsorption spectrum of the filtrate exhibits maxima and minima at the same wavelengths as that of a 1in 100,000solution of
USP Phytonadione RSin dehydrated alcohol concomitantly measured.
B:
The retention time of the major peak in the chromatogram of the Assay preparationcorresponds to that in the chromatogram of the Standard preparation,as obtained in the Assay.
Assay—
[NOTE—Use low-actinic glassware throughout the
Assay,and otherwise protect the solutions from light.
]
Mobile phase—
Prepare a suitable filtered and degassed mixture of dehydrated alcohol and water (95:5).
Standard preparation—
Prepare a solution of
USP Phytonadione RSin dehydrated alcohol having a known concentration of about 0.10mg per mL.
Assay preparation—
Weigh and finely powder not less than 20Tablets.Transfer a portion of the powdered Tablets,equivalent to about 5mg of phytonadione,to a 50-mLvolumetric flask,add 20mLof dehydrated alcohol,and shake by mechanical means for 15minutes.Dilute with dehydrated alcohol to volume,mix,and filter.
Chromatographic system
(see
Chromatography á621ñ)—The liquid chromatograph is equipped with a 254-nm detector and a 4-mm ×30-cm column that contains packing L1.The flow rate is about 1.5mLper minute.Chromatograph three replicate injections of the
Standard preparation,and record the peak responses as directed for
Procedure:the column efficiency determined from the analyte peak is not less than 915theoretical plates,the relative standard deviation is not more than 2.0%,and the tailing factor is not more than 2.0.
Procedure—
Separately inject equal volumes (about 10µL)of the
Standard preparationand the
Assay preparationinto the chromatograph,record the chromatograms,and measure the response for the major peak.Calculate the quantity,in mg,of C
31H
46O
2,in the portion of Tablets taken by the formula:
50C(rU/rS),
in which
Cis the concentration,in mg per mL,of
USP Phytonadione RSin the
Standard preparation,and
rUand
rSare the peak responses obtained from the
Assay preparationand the
Standard preparation,respectively.