Calcipotriene Ointment

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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1 DEFINITION 

To view the Notice from the Expert Committee that posted in conjunction with this accelerated revision

Calcipotriene and Betamethasone Dipropionate Ointment contains NLT 90.0% and NMT 114.0% of the labeled amount of calcipotriene (C₂₇H₄₀O₃) 

2 IDENTIFICATION 

A. The retention time of the major peak of the Sample solution corresponds to that of the Standard solution, as obtained in the Assay.

B. The UV spectrum of the major peak of the Sample solution corresponds to that of the Standard solution, as obtained in the Assay.

3 ASSAY 

Change to read:

Procedure

Protect solutions containing calcipotriene from light and air. Prepare the solutions containing calcipotriene NMT 1 h before use. 

Mobile phase: Methanol and water (70:30)

Buffer: 132 g/L of monobasic ammonium phosphate in water

Diluent: Methanol, Buffer, and water (700:3:297)

Standard stock solution: 0.1 mg/mL of USP Calcipotriene RS in Diluent. Sonicate if necessary.

Standard solution: 2 μg/mL of USP Calcipotriene RS prepared as follows. Transfer 5 mL of Standard stock solution into a 250-mL volumetric flask, add 50 mL of tetrahydrofuran, and dilute with Diluent to volume.

Sample stock solution: Nominally equivalent to 0.01 mg/mL of calcipotriene in tetrahydrofuran prepared as follows. Transfer Ointment equivalent to 0.25 mg of calcipotriene into a 25-mL volumetric flask. Add 15 mL of tetrahydrofuran and sonicate, with intermittent shaking, for 20 min in a cold water bath. Dilute with tetrahydrofuran to volume.

Sample solution: Nominally equivalent to 2 μg/mL of calcipotriene prepared as follows. Transfer 5 mL of the Sample stock solution into a suitable container. Add 20 mL of Diluent, mix, and sonicate for 10 min. Pass through a suitable filter of 0.45-μm pore size. Inject immediately after preparation.

Chromatographic system

(See Chromatography 〈621〉, System Suitability.)

Mode: LC

Detector: UV 264 nm. For Identi cation B, use a diode array detector in the range of 190–400 nm. 

Column: 4.6-mm × 15-cm; 3-μm packing L1

Flow rate: 1.0 mL/min

Injection volume: 50 μL

System suitability

Sample: Standard solution

Suitability requirements

Tailing factor: NMT 2.0

Relative standard deviation: NMT 2.0%

Analysis 

Samples: Standard solution and Sample solution 

Calculate the percentage of the labeled amount of calcipotriene (C₂₇H₄₀O₃) in the portion of Ointment taken:

Result = (r_U/r_S) x (C_S/C_U) × 100 

r_U = peak response of calcipotriene from the Sample solution

r_S = peak response of calcipotriene from the Standard solution

C_S = concentration of USP Calcipotriene RS in the Standard solution (mg/mL)

C_U = nominal concentration of calcipotriene in the Sample solution (mg/mL) 

Acceptance criteria: 90.0%–110.0%

Minimum Fill 〈755〉: Meets the requirements

4 IMPURITIES

Change to read:

Organic Impurities

Protect solutions containing calcipotriene from light and air. Prepare the Standard solution and the Sample solution NMT 1 h before use. Prepare the System suitability solution daily.

Mobile phase, Buffer, and Diluent: Prepare as directed in the Assay.

System suitability solution: 10.0 μg/mL of USP Calcipotriene RS and 0.1 μg/mL of USP Calcipotriene Related Compound C RS in Diluent

Standard stock solution: 1.0 μg/mL of USP Calcipotriene RS in Diluent

Standard solution: 0.1 μg/mL of USP Calcipotriene RS prepared as follows. Transfer 1.0 mL of the Standard stock solution into a 10-mL volumetric flask, add 1.0 mL tetrahydrofuran, and dilute with Diluent to volume.

Sensitivity solution: 0.01 μg/mL of USP Calcipotriene RS prepared as follows. Transfer 1.0 mL of the Standard solution into a 10-mL volumetric flask, add 1.0 mL tetrahydrofuran, and dilute with Diluent to volume.

Sample solution: Nominally equivalent to 0.01 mg/mL of calcipotriene prepared as follows. Transfer Ointment equivalent to 0.1 mg of calcipotriene into a glass-stoppered test tube, and add 1 mL of tetrahydrofuran. Sonicate for 20 min with intermittent shaking. Add 9 mL of Diluent, and sonicate for 5 min. Shake the test tube vigorously, and then place it in a beaker containing ice cold water for 2–3 min. Pass the liquid portion through a nylon filter of 0.45-μm pore size, and discard the first few milliliters of the solution.

Chromatographic system

(See Chromatography 〈621〉, System Suitability.)

Mode: LC

Detector: UV 264 nm

Column: 4.6-mm × 15-cm; 3-μm packing L1

Flow rate: 1.0 mL/min

Injection volume: 100 μL

Run time: NLT 1.25 times of retention time of the calcipotriene peak

System suitability

Samples: System suitability solution, Standard solution, and Sensitivity solution 

Suitability requirements 

Resolution: NLT 1.0 between calcipotriene and calcipotriene related compound C, System suitability solution 

Tailing factor: 0.8–1.4 for calcipotriene, Sample solution 

Relative standard deviation: NMT 2.0% for the peak height ratio of calcipotriene to methyltestosterone, Standard solution Analysis 

Samples: Standard solution and Sample solution 

[Note-The relative retention times for betamethasone 17-propionate, betamethasone 21-propionate, methyl Testosterone, betamethasone dipropionate, and calcipotriene (plus pre-calcipotriene) are about 0.28, 0.28, 0.38, 0.52, and 1.0, respectively. The controlled isomerization of calcipotriene is carried out at 50° producing a mixture of calcipotriene and the isomer (pre-calcipotriene), which is eluted as a single peak.] Calculate the percentage of the labeled amount of calcipotriene (C₂₇H₄₀O₃) in the portion of Ointment taken:  

Result =(R_U/R_S) × (C_S/C_U) × 100 

R_U = peak height ratio of calcipotriene (plus pre-calcipotriene) to Methyltestosterone from the Sample solution  

R_S = peak height ratio of calcipotriene (plus pre-calcipotriene) to methyltestosterone from the Standard solution 

C_S = concentration of USP Calcipotriene RS in the Standard solution (mg/mL) 

C_U = nominal concentration of calcipotriene in the Sample solution (mg/mL) 

Acceptance criteria: 90.0%–114.0% 

5 IMPURITIES

Organic Impurities: Betamethasone Dipropionate 

[Note-Protect solutions containing betamethasone dipropionate from light.] 

Buffer: 8.9 g/L of sodium phosphate dibasic dihydrate in water. Adjust with 1 M phosphoric acid TS to a pH of 7.0 ± 0.1. Mobile phase: Acetonitrile and Buffer (50:55) 

Sample solution: Suspend 1.0 g of Ointment in 10 mL of n-heptane by heating on a steam bath until the Ointment melts. Add 8.0 mL of Mobile phase and shake vigorously for 15 min. Clear the lower layer by centrifugation in glass centrifugation tubes and use the clear lower layer. 

Chromatographic system 

(See Chromatography 〈621〉, System Suitability.) 

Mode: LC 

Detector: UV 240 nm. [Note-It is recommended to use a diode array detector in the range of 200–320 nm. It makes it easier to identify the betamethasone dipropionate related impurities in the presence of calcipotriene, calcipotriene related impurities, and any excipients present.] 

Column: 4-mm × 12.5-cm; 5-µm packing L1 

Flow rate: 2 mL/min 

Injection volume: 20 µL 

Run time: NLT 3.5 times the retention time of betamethasone dipropionate 

System suitability 

Sample: Sample solution 

[Note-See Table 1 for the relative retention times.] 

Suitability requirements 

Resolution: NLT 1.2 between calcipotriene, System suitability solution

Relative standard deviation: NMT 5.0%, Standard solution

Signal-to-noise ratio: NLT 10, Sensitivity solution

Analysis 

Samples: Standard solution and Sample solution

Calculate the percentage of each individual impurity in the portion of Ointment taken:

Result = (r_U/r_S) x (C_S/C_U) x× 100 

r_U = peak response of any impurity from the Sample solution

r_S = peak response of calcipotriene from the Standard solution

C_S = concentration of USP Calcipotriene RS in the Standard solution (mg/mL)

C_U = nominal concentration of calcipotriene in the Sample solution (mg/mL) 

Acceptance criteria: See Table 1. The reporting threshold is 0.1%.

Table 1

^a(5Z,7Z,22E,24S)-24-Cyclopropyl-9,10-secochola-5,7,10(19),22-tetraene-1α,3β,24-triol.
^b(5E,7E,22E,24S)-24-Cyclopropyl-9,10-secochola-5,7,10(19),22-tetraene-1α,3β,24-triol.
^c(5Z,7E,22E,24R)-24-Cyclopropyl-9,10-secochola-5,7,10(19),22-tetraene-1α,3β,24-triol.

^dDoes not apply if propylene glycol ethers of calcipotriene are present in the manufacturing process. Calculate the total impurities in the test for Limit of Propylene Glycol Ethers of Calcipotriene.

Add the following:

Limit of Propylene Glycol Ethers of Calcipotriene (if present)

Protect solutions containing calcipotriene from light and air. Prepare the solutions containing calcipotriene NMT 1 h before use.

Solution A: Add 70 mL of phosphoric acid in 1000 mL of water

Solution B: 1.32 g/L of dibasic ammonium phosphate in water

Solution C: Adjust Solution B with Solution A to a pH of 6.0 ± 0.1.

Solvent for extraction: Acetonitrile, methanol, and Solution B (20:50:30)

Mobile phase: Acetonitrile, methanol, and Solution C (20:50:30)

System suitability stock solution: 0.05 mg/mL of USP Calcipotriene RS in Solvent for extraction

System suitability solution: 1.0 μg/mL of USP Calcipotriene RS prepared as follows. Transfer 1.0 mL of System suitability stock solution and 15 mL of water into a 50-mL volumetric flask and dilute with Solvent for extraction to volume.

Sample stock solution: Mix all contents of two tubes of Ointment. Weigh 2 g into a glass with a screw cap, and suspend the Ointment in 8 mL of n-heptane by gentle heating on a steam bath. Add 8 mL of Solvent for extraction, and shake vigorously for 15 min. Centrifuge to separate the two phases if needed.

Sample solution: Mix 3.5 mL of lower phase of the Sample stock solution with 1.5 mL of water. Pass the solution through glass fiber filter,1 and use the filtrate for analysis.

Chromatographic system

(See Chromatography 〈621〉, System Suitability.)

Mode: LC

Detector: UV 265 nm

Column: 4-mm × 12.5-cm; 4-μm packing L1

Flow rate: 1.2 mL/min

Injection volume: 500 μL

Run time: NLT 3 times of retention time of the calcipotriene peak

System suitability

Sample: System suitability solution

Suitability requirements

Relative standard deviation: NMT 10.0%

Analysis

Sample: Sample solution

The peaks of propylene glycol ethers of calcipotriene are the peaks in the range of relative retention times between 1.4 and 3.0 measured with respect to the calcipotriene peak.

Calculate the percentage of the propylene glycol ethers of calcipotriene in the portion of Ointment taken:

Result = (r_U/r_T) × 100 

r_U = peak response of the propylene glycol ethers of calcipotriene from the Sample solution

r_T = sum of the peak responses from the Sample solution

Acceptance criteria

Propylene glycol ethers of calcipotriene: NMT 6.0%
Total impurities: NMT 10.0%. The total impurities are the sum of all impurities in Table 1 and propylene glycol ethers of calcipotriene.

6 SPECIFIC TESTS 

Microbial Enumeration Tests 〈61〉 and Tests for Specified Microorganisms〈62〉: The total aerobic microbial count is NMT 10² cfu/g. The total combined molds and yeasts count is NMT 10¹ cfu/g. It meets the requirements of the tests for absence of Staphylococcus aureus and Pseudomonas aeruginosa. 

Delete the following:

pH 〈791〉

7 ADDITIONAL REQUIREMENTS 

Packaging and Storage: Preserve in well-closed containers, and store at controlled room temperature. Do not freeze.

USP Reference Standards 〈11〉 

USP Calcipotriene RS 

USP Calcipotriene Related Compound C RS 

(5E,7E,22E,24S)-24-Cyclopropyl-9,10-secochola-5,7,10(19),22-tetraene-1α,3β,24-triol. 

C₂₇H₄₀O₃   412.61 

1 A suitable filter is available as GD/A-grade glass fiber filter from Whatman