Assay—
[NOTE—Use low-actinic glassware throughout this assay, and otherwise protect the solutions from exposure to light.
]
Mobile phase—
Prepare a suitable degassed mixture of dehydrated alcohol and water (95:5).
Standard preparation—
Dissolve an accurately weighed quantity of
USP Phytonadione RS in
Mobile phase to obtain a solution having a known concentration of about 1 mg per mL. Pipet 1 mL of this solution into a 10-mL volumetric flask, dilute with
Mobile phase to volume, and mix to obtain a
Standard preparation having a known concentration of about 0.1 mg per mL.
Assay preparation 1 (containing 10 mg or more of phytonadione per mL)—
Pipet a volume of Injectable Emulsion, equivalent to 10 mg of phytonadione, into a 10-mL volumetric flask, dilute with Mobile phase to volume, and mix. Pipet 1 mL of this solution into a 10-mL volumetric flask, dilute with Mobile phase to volume, and mix.
Assay preparation 2 (containing less than 10 mg of phytonadione per mL)—
Pipet a volume of Injectable Emulsion, equivalent to 1 mg of phytonadione, into a 10-mL volumetric flask, dilute with Mobile phase to volume, and mix.
Chromatographic system (see Chromatography 
621
)—
The liquid chromatograph is equipped with a 254-nm detector and a 4-mm × 25-cm column that contains packing L1. The flow rate is about 0.7 mL per minute. Chromatograph five replicate injections of the
Standard preparation, and record the peak responses as directed for
Procedure: the relative standard deviation is not more than 1.5%.
Procedure—
Separately inject equal volumes (about 10 µL) of the
Standard preparation and the appropriate
Assay preparation into the chromatograph, record the chromatograms, and measure the peak response for the major peak. Calculate the quantity, in mg, of C
31H
46O
2 in each mL of the Injectable Emulsion taken by the formula:
D(C/V)(rU / rS),
in which
D is 100 if the Injectable Emulsion contains 10 mg or more of phytonadione per mL, or 10 if the Injectable Emulsion contains less than 10 mg of phytonadione per mL;
C is the concentration, in mg per mL, of
USP Phytonadione RS in the
Standard preparation; V is the volume, in mL, of Injectable Emulsion taken; and
rU and
rS are the peak responses of phytonadione obtained from the appropriate
Assay preparation and the
Standard preparation, respectively.
Phytonadione Injection