Z isomer 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
rZ is the peak area of the (
Z)-phytonadione isomer peak and
rE is 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 phase to obtain a solution having a concentration of 2.5 mg per mL.
Standard preparation—
Transfer about 60 mg of
USP Phytonadione RS, accurately weighed, to a 50-mL volumetric flask, add 20 mL of
Mobile phase, mix, dilute with
Mobile phase to volume, and again mix. Pipet 4 mL of the resulting solution into a 50-mL volumetric flask, dilute with
Mobile phase to volume, and mix. Pipet 10 mL of this solution and 7 mL of
Internal standard solution into a 25-mL volumetric flask, dilute with
Mobile phase to volume, and mix.
Assay preparation—
Prepare as directed under Standard preparation, using Phytonadione instead of the Reference Standard.
Chromatographic system
(see
Chromatography 
621
)—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 1 mL per 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 50 µL) of the
Standard preparation and the
Assay preparation into the chromatograph, record the chromatograms, and measure the responses for the major peaks. The relative retention times are about 0.7 for the internal standard, 0.9 for (
Z)-phytonadione, and 1.0 for (
E)-phytonadione. Calculate the quantity, in mg, of C
31H
46O
2 in the portion of Phytonadione taken by the formula:
1.56C(RU / RS),
in which
C is the concentration, in µg per mL, of
USP Phytonadione RS in the
Standard preparation, and
RU and
RS are the relative peak response ratios for the
Assay preparation and the
Standard preparation, respectively. Calculate
RU and
RS by the formula:
(response for the (Z)-phytonadione peak + response for the (E)-phytonadione peak) / response for the internal standard peak.
;)
