Phytonadione
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C31H46O2 450.70

1,4-Naphthalenedione,2-methyl-3-(3,7,11,15-tetramethyl-2-hexadecenyl)-,[R-[R*,R*-(E)]]-.
Phylloquinone [84-80-0].
»Phytonadione is a mixture of Eand Zisomers containing not less than 97.0percent and not more than 103.0percent of C31H46O2.It contains not more than 21.0percent of the Zisomer.
Packaging and storage— Preserve in tight,light-resistant containers.
Identification—
A: Infrared Absorption á197Fñ.
Solution: 10µg per mL.
Medium:n -hexane.
Absorptivities at 248nm do not differ by more than 3.0%.
Refractive index á831ñ: between 1.523and 1.526.
Reaction— A1in 20solution of it in dehydrated alcohol is neutral to litmus.
Limit of menadione— Mix about 20mg with 0.5mLof a mixture of equal volumes of 6Nammonium hydroxide and alcohol,then add 1drop of ethyl cyanoacetate,and shake gently:no purple or blue color is produced.
Zisomer content— [NOTE—Protect solutions containing Phytonadione from exposure to light.]
Mobile phase,Internal standard solution,Assay preparation,Chromatographic system,and Procedure— Proceed as directed in the Assay,except to calculate the percentage of Zisomer taken by the formula:
100rZ/(rZ+rE),
in which rZis the peak area of the (Z)-phytonadione isomer peak and rEis the peak area of the (E)-phytonadione isomer peak obtained from the Assay preparation.
Assay— [NOTE—Protect solutions containing Phytonadione from exposure to light.]
Mobile phase— Prepare a filtered and degassed solution of n-hexane and n-amyl alcohol (2000:1.5).
Internal standard solution— Dissolve cholesteryl benzoate in Mobile phaseto obtain a solution having a concentration of 2.5mg per mL.
Standard preparation— Transfer about 60mg of USP Phytonadione RS,accurately weighed,to a 50-mLvolumetric flask,add 20mLof Mobile phase,mix,dilute with Mobile phaseto volume,and again mix.Pipet 4mLof the resulting solution into a 50-mLvolumetric flask,dilute with Mobile phaseto volume,and mix.Pipet 10mLof this solution and 7mLof Internal standard solutioninto a 25-mLvolumetric flask,dilute with Mobile phaseto volume,and mix.
Assay preparation— Prepare as directed under Standard preparation,using Phytonadione instead of the Reference Standard.
Chromatographic system (see Chromatography á621ñ)—The liquid chromatographic is equipped with a 254-nm detector and a 4.6-mm ×25-cm column that contains packing L3.The flow rate is about 1mLper minute.Chromatograph replicate injections of the Standard preparation,and record the peak responses as directed for Procedure:the relative standard deviation is not more than 2.0%,and the resolution,R,between (Z)-phytonadione and (E)-phytonadione is not less than 1.5.
Procedure— Separately inject equal volumes (about 50µL)of the Standard preparationand the Assay preparationinto the chromatograph,record the chromatograms,and measure the responses for the major peaks.The relative retention times are about 0.7for the internal standard,0.9for (Z)-phytonadione,and 1.0for (E)-phytonadione.Calculate the quantity,in mg,of C31H46O2in the portion of Phytonadione taken by the formula:
1.56C(RU/RS),
in which Cis the concentration,in µg per mL,of USP Phytonadione RSin the Standard preparation,and RUand RSare the relative peak response ratios for the Assay preparationand the Standard preparation,respectively.Calculate RUand RSby the formula:
(response for the (Z)-phytonadione peak +response for the (E)-phytonadione peak)/response for the internal standard peak.
Auxiliary Information— Staff Liaison:Lawrence Evans,III,Ph.D.,Scientist
Expert Committee:(DSN)Dietary Supplements:Non-Botanicals
USP28–NF23Page 1557
Phone Number:1-301-816-8389