Phytonadione

(fye toe'' na dye' one).

C31H46O2    450.70
1,4-Naphthalenedione, 2-methyl-3-(3,7,11,15-tetramethyl-2-hexadecenyl)-, [R-[R*,R*-(E)]]-;
Phylloquinone

[84-80-0].

DEFINITION

Phytonadione is a mixture of E- and Z-isomers containing NLT 97.0% and NMT 103.0% of phytonadione isomers (C31H46O2). It contains NMT 21.0% of the Z-isomer.

IDENTIFICATION

• A. Infrared Absorption

197F

• B. Ultraviolet Absorption

197U

Analytical wavelength: 248 nm

Sample solution: 10 µg/mL in n-hexane

Acceptance criteria: Meets the requirements. Absorptivities do not differ by more than 3.0%.

ASSAY

• Procedure

[Note—Protect solutions containing phytonadione from exposure to light. ]

Mobile phase: n-Hexane and n-amyl alcohol (2000: 1.5)

Internal standard solution: 2.5 mg/mL of cholesteryl benzoate in Mobile phase

Standard solution: Prepare a 96-µg/mL solution of USP Phytonadione RS in Mobile phase. Pipet 10 mL of this solution and 7 mL of Internal standard solution into a 25-mL volumetric flask, and dilute with Mobile phase to volume.

Sample solution: Prepare as directed for the Standard solution, using Phytonadione instead of USP Phytonadione RS.

Chromatographic system

(See Chromatography

621

, System Suitability.)

Mode: LC

Detector: UV 254 nm

Column: 4.6-mm × 25-cm; packing L3

Flow rate: 1 mL/min

Injection size: 50 µL

System suitability

Sample: Standard solution

[Note—The relative retention times for the internal standard, (Z)-phytonadione, and (E)-phytonadione are 0.7, 0.9, and 1.0, respectively. ]

Suitability requirements

Resolution: NLT 1.5 between (Z)-phytonadione and (E)-phytonadione

Relative standard deviation: NMT 2.0% for the ratios of the sum of peak areas of (Z)-phytonadione and (E)-phytonadione to the peak area of the internal standard

Analysis

Samples: Standard solution and Sample solution

Calculate the percentage of phytonadione (C31H46O2) in the portion of Phytonadione taken:

Result = (RU/RS) × (CS/CU) × 100

RU	=	= internal standard ratio (sum of peak areas of (Z)-phytonadione and (E)-phytonadione/peak area of the internal standard) from the Sample solution
RS	=	= internal standard ratio (sum of peak areas of (Z)-phytonadione and (E)-phytonadione/peak area of the internal standard) from the Standard solution
CS	=	= concentration of USP Phytonadione RS in the Standard solution (µg/mL)
CU	=	= concentration of Phytonadione in the Sample solution (µg/mL)

Acceptance criteria: 97.0%–103.0%

OTHER COMPONENTS

• Z-Isomer Content

[Note—Protect solutions containing phytonadione from exposure to light. ]

Mobile phase, Internal standard solution, Sample solution, Chromatographic system, and Analysis: Proceed as directed in the Assay, except calculate the percentage of Z-isomer in the portion of Phytonadione taken:

Result = [rZ/(rZ + rE)] × 100

rZ	=	= peak area of the (Z)-phytonadione isomer from the Sample solution
rE	=	= peak area of the (E)-phytonadione isomer from the Sample solution

Acceptance criteria: NMT 21.0%

IMPURITIES

• Limit of Menadione

Sample: 20 mg of Phytonadione

Analysis: Mix the Sample with 0.5 mL of a mixture of 6 N ammonium hydroxide and alcohol (1:1). Add 1 drop of ethyl cyanoacetate, and shake gently.

Acceptance criteria: No purple or blue color is produced.

SPECIFIC TESTS

• Refractive Index

831

: 1.523–1.526

• Reaction: A 50-mg/mL solution of Phytonadione in dehydrated alcohol is neutral to litmus.

ADDITIONAL REQUIREMENTS

• Packaging and Storage: Preserve in tight, light-resistant containers.

• USP Reference Standards

USP Phytonadione RS

Auxiliary Information— Please check for your question in the FAQs before contacting USP.

Topic/Question	Contact	Expert Committee
Monograph	Huy T. Dinh, M.S. Scientific Liaison 1-301-816-8594	(DS2010) Monographs - Dietary Supplements
Reference Standards	RS Technical Services 1-301-816-8129 rstech@usp.org

USP35–NF30 Page 4318

Chromatographic Column—

PHYTONADIONE

Chromatographic columns text is not derived from, and not part of, USP 35 or NF 30.