Calcipotriene and Betamethasone Dipropionate 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 

Calcipotriene and Betamethasone Dipropionate Ointment contains NLT 90.0% and NMT 114.0% of the labeled amount of calcipotriene (C₂₇H₄₀O₃) and NLT 90.0% and NMT 110.0% of the labeled amount of betamethasone dipropionate (C₂₈H₃₇FO₇) in a suitable ointment base. 

2 IDENTIFICATION 

A. The retention time of the betamethasone dipropionate peak from the Sample solution corresponds to that of the Standard solution, as obtained in the Assay for Betamethasone Dipropionate. 

B. The retention time of the calcipotriene peak from the Sample solution corresponds to that of the Standard solution, as obtained in the Assay for Calcipotriene. 

C. The UV spectrum of the betamethasone dipropionate peak from the Sample solution corresponds to that of the Standard solution, as obtained in the Assay for Betamethasone Dipropionate. 

D. The UV spectrum of the calcipotriene peak from the Sample solution corresponds to that of the Standard solution, as obtained in the Assay for Calcipotriene. 

3 ASSAY 

Betamethasone Dipropionate 

[Note-Protect solutions containing betamethasone dipropionate and beclomethasone dipropionate from light.] Mobile phase: Acetonitrile and water (50:55) 

Diluent: Acetonitrile and water (60:25) 

Internal standard solution: 0.4 mg/mL of USP Beclomethasone Dipropionate RS in Diluent 

Standard stock solution: 0.87 mg/mL of USP Betamethasone Dipropionate RS in Diluent 

Standard solution: 0.012 mg/mL of USP Betamethasone Dipropionate RS from the Standard stock solution prepared as follows. Dilute 1.0 mL of the Standard stock solution with 2.0 mL of Internal standard solution followed by 22.0 mL of Diluent and 50.0 mL of Mobile phase.

Sample solution: Nominally 0.012 mg/mL of betamethasone dipropionate from Ointment prepared as follows. Suspend 0.675 g of Ointment in 25 mL of n-heptane by heating on a steam bath until the Ointment melts. Add 1.0 mL of Internal standard solution and 11.5 mL of Diluent and shake for 15 min. Add 25 mL of Mobile phase and clear by centrifugation, if necessary, using glass centrifuge tubes. Use the clear lower layer. 

Chromatographic system 

(See Chromatography 〈621〉, System Suitability.) 

Mode: LC 

Detector: UV 240 nm. For Identification C, use a diode array detector in the range of 200–400 nm. 

Column: 4-mm × 7.5-cm; 4-µm packing L1 

Flow rate: 1.5 mL/min 

Injection volume: 20 µL 

Run time: NLT 1.5 times the retention time of betamethasone dipropionate 

System suitability 

Samples: Standard solution and Sample solution 

[Note-The relative retention times for calcipotriene, betamethasone dipropionate, and beclomethasone dipropionate are about 0.78, 1.0, and 1.3, respectively.] 

Suitability requirements 

Resolution: NLT 1.0 between calcipotriene and betamethasone dipropionate; NLT 2.5 between betamethasone dipropionate and beclomethasone dipropionate, Sample solution 

Tailing factor: 0.8–1.4 for betamethasone dipropionate, Sample solution 

Relative standard deviation: NMT 2.0% for the peak height ratio of betamethasone dipropionate to beclomethasone dipropionate, Standard solution 

Analysis 

Samples: Standard solution and Sample solution 

Calculate the percentage of the labeled amount of betamethasone dipropionate (C₂₈H₃₇FO₇) in the portion of Ointment taken: 

Result = (R_U/R_S) × (C_S/C_U) × 100 

R_U = peak height ratio of betamethasone dipropionate to beclomethasone dipropionate from the Sample solution 

R_S = peak height ratio of betamethasone dipropionate to beclomethasone dipropionate from the Standard solution 

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

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

Acceptance criteria: 90.0%–110.0% 

Calcipotriene

[Note-Protect solutions containing calcipotriene from light.] 

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

Buffer: Solution A, adjusted with 1 M phosphoric acid TS to a pH of 6.0 

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

Diluent: Methanol and Solution A (70:30) 

Internal standard solution: 0.1 mg/mL of USP Methyltestosterone RS in Diluent 

System suitability solution: 0.0025 mg/mL each of USP Calcipotriene RS and USP Calcipotriene Related Compound C RS in acetonitrile, methanol, and Solution A (20:50:30) 

Standard stock solution: 0.05 mg/mL of USP Calcipotriene RS in Diluent 

Standard solution: 0.0025 mg/mL of USP Calcipotriene RS from the Standard stock solution prepared as follows. Dilute 1.0 mL of the Standard stock solution with 1.0 mL of Internal standard solution followed by 18.0 mL of Diluent. Transfer a portion to a glass test tube with a screw cap and heat at 50° for 15–18 h. Cool to room temperature in a water bath. Prepare this solution simultaneously with the Sample solution. 

Sample solution: Nominally 0.0025 mg/mL of calcipotriene from Ointment prepared as follows. Suspend 1.0 g of Ointment in 10 mL of n heptane by heating on a steam bath until the Ointment melts. Add 1.0 mL of Internal standard solution and 19.0 mL of Diluent and shake for 30 min. If necessary, clear the lower layer by centrifugation using glass centrifuge tubes. Transfer a portion of the lower layer to a glass test tube with a screw cap and heat at 50° for 15–18 h. Cool to room temperature in a water bath. Prepare this solution simultaneously with the Standard solution. 

Chromatographic system 

(See Chromatography 〈621〉, System Suitability.) 

Mode: LC 

Detector: UV 264 nm. For Identification D, use a diode array detector in the range of 200–400 nm. 

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

Flow rate: 2 mL/min 

Injection volume: 50 µL 

Run time: NLT 1.2 times the retention time of calcipotriene 

System suitability 

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

[Note-The relative retention times for calcipotriene related compound C and calcipotriene are about 0.9 and 1.0, respectively.]

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% 

4 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 1.0 between betamethasone dipropionate and calcipotriene 

Relative standard deviation: NMT 5.0% for betamethasone dipropionate

Analysis 

Sample: Sample solution

Calculate the percentage of each betamethasone dipropionate related degradation product in the portion of Ointment taken: 

Result = (r_U/r_T) × 100 

r_U = peak response of each betamethasone dipropionate related degradation product 

r_T = sum of the peak responses of betamethasone dipropionate and all betamethasone dipropionate related degradation products 

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

^a 9-Fluoro-11β,21-dihydroxy-16β-methyl-3,20-dioxopregna-1,4-diene-17-yl propionate. 

^b 9-Fluoro-11β,17-dihydroxy-16β-methyl-3,20-dioxopregna-1,4-diene-21-yl propionate. 

^c(5E,6E,22E,24S)-24-Cyclopropyl-9,10-secochola-5(10),6,22-triene-1α,3β,24-triol. 

^d Included for Identification purposes only. 

Organic Impurities: Calcipotriene 

[Note-Protect solutions containing calcipotriene from light.] 

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

Buffer: Solution A, adjusted with 1 M phosphoric acid TS to a pH of 6.0 ± 0.1 

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

Diluent: Acetonitrile, methanol, and Solution A (20:50:30) 

System suitability stock solution: 0.05 mg/mL each of USP Calcipotriene RS and USP Calcipotriene Related Compound C RS in Diluent System suitability solution: 1 µg/mL each of USP Calcipotriene RS and USP Calcipotriene Related Compound C RS from the System suitability stock solution prepared as follows. To 10.0 mL of the System suitability stock solution add 340 mL of Diluent and 150 mL of water. Sample solution: Suspend 2.0 g of Ointment in 8 mL of n-heptane by heating on a steam bath until the Ointment melts. Add 8.0 mL of Diluent and shake vigorously for 15 min. Mix 3.5 mL of the lower phase with 1.5 mL of water, and clear by centrifugation using glass centrifuge tubes, if necessary. Filter through a suitable glass paper lter. 

Chromatographic system 

(See Chromatography 〈621〉, System Suitability.) 

Mode: LC 

Detector: UV 264 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 calcipotriene related impurities in the presence of betamethasone dipropionate, betamethasone dipropionate related impurities, and any excipients present.] 

Column: 4-mm × 7.5-cm and 4-mm × 12.5-cm in series; 4-µm packing L1 

Flow rate: 1 mL/min 

Injection volume: 500 µL 

Run time: NLT 2 times the retention time of calcipotriene 

System suitability 

Sample: System suitability solution Suitability requirements 

Resolution: NLT 1.8 between calcipotriene related compound C and calcipotriene 

Relative standard deviation: NMT 5.0% each for calcipotriene related compound C and calcipotriene 

Analysis 

Sample: Sample solution 

Calculate the percentage of each calcipotriene related degradation product in the portion of Ointment taken: 

Result = (r_U/r_T) × 100 

r_U = peak response of each calcipotriene related degradation product 

r_T = sum of the peak responses of calcipotriene and all calcipotriene related degradation products 

Acceptance criteria: See Table 2. The reporting threshold is 0.1%. 

Table 2 

^a  Included for Identification purposes only.

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

5 PERFORMANCE TESTS 

Minimum Fill 〈755〉: Meets the requirements 

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. 

7 ADDITIONAL REQUIREMENTS 

Packaging and Storage: Preserve in well-closed containers, and store at controlled room temperature. 

USP Reference Standards 〈11〉 

USP Beclomethasone Dipropionate RS USP Betamethasone Dipropionate RS

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 

USP Methyltestosterone RS