Phytonadione

This article is compiled based on the United States Pharmacopeia (USP) – 2025 Edition

Issued and maintained by the United States Pharmacopeial Convention (USP)

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2S (USP41)

C₃₁H₄₆O₂       450.70

2-Methyl-3-(3,7,11,15-tetramethylhexadec-2-en-1-yl)naphthalene-1,4-dione;_{2S (USP41)}

Phylloquinone CAS RN®: 81818-54-4._{2S (USP41)}

1 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.

2 IDENTIFICATION

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A. Spectroscopic Identification Tests 〈197〉, Infrared Spectroscopy: 197F (CN 1-May-2020)

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B. Spectroscopic Identification Tests 〈197〉, Ultraviolet-Visible Spectroscopy: 197U (CN 1-May-2020)

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%.

3 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 ask, and dilute with Mobile phase to volume.

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

3.1 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 volume: 50 µL

3.2 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

3.3 Analysis

Samples: Standard solution and Sample solution

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

Result = (R_U /R_S ) × (C_S /C_U) × 100

R_U = internal standard ratio [sum of the peak areas of (Z)-phytonadione and (E)-phytonadione/peak area of the internal standard] from the Sample solution

R_S = internal standard ratio [sum of the peak areas of (Z)-phytonadione and (E)-phytonadione/peak area of the internal standard] from the Standard solution

C_S = concentration of USP Phytonadione RS in the Standard solution (µg/mL)

C_U = concentration of Phytonadione in the Sample solution (µg/mL)

Acceptance criteria: 97.0%–103.0% of phytonadione isomers

4 OTHER COMPONENTS

Z-Isomer Content

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

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

Result = [r_Z /(r_Z + r_E )] × 100

r_Z = peak area of the (Z)-phytonadione isomer from the Sample solution

r_E = peak area of the (E)-phytonadione isomer from the Sample solution

Acceptance criteria: NMT 21.0%

5 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.

6 SPECIFIC TESTS

Refractive Index 〈831〉: 1.523–1.526

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

7 ADDITIONAL REQUIREMENTS

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Packaging and Storage: Preserve in tight, light-resistant containers. Store in a cool place._{2S (USP41)}

USP Reference Standards 〈11〉

USP Phytonadione RS