Zoledronic Acid

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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C₅H₁₀N₂O₇P₂ · H₂O 290.10

Phosphonic acid, [1-hydroxy-2-(1H-imidazol-1-yl)ethylidene]bis-, monohydrate;

(1-Hydroxy-2-imidazol-1-ylethylidene)diphosphonic acid, monohydrate CAS RN®: 165800-06-6.

Anhydrous CAS RN®: 118072-93-8. 272.09

1 DEFINITION

Zoledronic acid contains NLT 98.0% and NMT 102.0% of zoledronic acid (C₅H₁₀N₂O₇P₂), calculated on the anhydrous basis.

2 IDENTIFICATION

A. Spectroscopic Identification Tests 〈197〉, Infrared Spectroscopy: 197K or 197A

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

3 ASSAY

Procedure

Perchloric acid solution: Carefully add 10 mL of perchloric acid to 1 L of water, then add 2 mL of phosphoric acid, and mix.

Buffer: 0.037 g/L of edetate disodium and 10.8 g/L of octanesulfonic acid sodium salt in Perchloric acid solution

Mobile phase: Acetonitrile and Buffer (4:96)

System suitability solution: 2 μg/mL each of USP Imidazole RS, USP Zoledronic Acid Related Compound A RS, and sodium nitrate; 5 μg/mL of USP Zoledronic Acid Related Compound B RS; and 1.2 mg/mL of USP Zoledronic Acid RS in Mobile phase

Standard solution: 1 mg/mL of USP Zoledronic Acid RS in Mobile phase. Sonicate with occasional shaking for about 15 min to achieve complete dissolution.

Sample solution: 1 mg/mL of Zoledronic Acid in Mobile phase. Sonicate with occasional shaking for about 15 min to achieve complete dissolution.

Chromatographic system

(See Chromatography 〈621〉, System Suitability.)

[Note - Plastic HPLC vials are recommended.]

Preconditioning of the instrument: Rinse the instrument without a column with a mixture of acetic acid and water (25:75) at 5 mL/min for 20 min. Then, rinse with water at 5 mL/min for about 2 h.

Preconditioning of the column: Rinse with Mobile phase at 0.6 mL/min for about 1 h. During the rinsing, inject the Sample solution 10–15 times, applying a run time of about 3 min for each injection.

Mode: LC

Detector: UV 215 nm

Column: 4.6-mm × 25-cm; 5-μm packing L11

Flow rate: 0.6 mL/min

Injection volume: 5 μL

Run time: NLT 3 times the retention time of zoledronic acid

System suitability

Samples: System suitability solution and Standard solution

Suitability requirements

Resolution: NLT 1.0 between the nitrate and zoledronic acid related compound B peaks, System suitability solution

Tailing factor: NMT 1.4 for the zoledronic acid peak, System suitability solution

Relative standard deviation: NMT 0.73%, Standard solution

Analysis

Samples: Standard solution and Sample solution

Calculate the percentage of zoledronic acid (C₅H₁₀N₂O₇P₂) in the portion of Zoledronic Acid taken:

Result = (r_U/r_S) × (C_S/C_U) × 100

r_U = peak response of zoledronic acid from the Sample solution

r_S = peak response of zoledronic acid from the Standard solution

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

C_U = concentration of Zoledronic Acid in the Sample solution (mg/mL)

Acceptance criteria: 98.0%–102.0% on the anhydrous basis

4 IMPURITIES

Change to read:

Organic Impurities

Perchloric acid solution, Buffer, Mobile phase, and System suitability solution: Prepare as directed in the Assay.

Standard stock solution: 0.4 mg/mL of USP Zoledronic Acid RS in Mobile phase. Sonicate with occasional shaking for about 15 min to achieve complete dissolution.

Sensitivity solution: 1 μg/mL of USP Zoledronic Acid RS in Mobile phase from Standard stock solution

Standard solution: 0.002 mg/mL of USP Zoledronic Acid RS in Mobile phase from Standard stock solution

Sample solution: 2 mg/mL of Zoledronic Acid in Mobile phase. Sonicate with occasional shaking for about 30 min to achieve complete dissolution.

Chromatographic system: Proceed as directed in the Assay, except for Injection volume and Run time.

Injection volume: 10 μL

Run time: NLT 5 times the retention time of zoledronic acid

System suitability

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

Suitability requirements

Resolution: NLT 1.0 between the nitrate and zoledronic acid related compound B peaks, System suitability solution

Tailing factor: NMT 1.4 for the zoledronic acid peak, Standard 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 any individual impurity in the portion of Zoledronic Acid taken:

Result = (r_U/r_S) × (C_S/C_U) × (1/F) x 100

r_U = peak response of any individual impurity (ERR 1-Mar-2022) from the Sample solution

r_S = peak response of zoledronic acid from the Standard solution

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

C_U = concentration of Zoledronic Acid in the Sample solution (mg/mL)

F = relative response factor (see Table 1)

Acceptance criteria: See Table 1. The reporting threshold is 0.05%.

Table 1

Limit of Phosphate and Phosphite (if present)

Mobile phase: Add 0.47 mL of formic acid to 2.5 L of water and adjust with 2 N sodium hydroxide to a pH of 3.5.

Phosphoric acid stock solution: 0.65 mg/mL of phosphoric acid in Mobile phase

Phosphorus acid stock solution: 0.50 mg/mL of phosphorous acid in Mobile phase

System suitability solution: 13 μg/mL of phosphoric acid from Phosphoric acid stock solution, 10 μg/mL of phosphorous acid from Phosphorus acid stock solution, and 1 mg/mL of USP Zoledronic Acid RS in Mobile phase

Standard solution: 0.013 mg/mL of phosphoric acid from Phosphoric acid stock solution and 10 μg/mL of phosphorous acid from Phosphorus acid stock solution

Sample solution: 1 mg/mL of Zoledronic Acid in Mobile phase

Chromatographic system

(See Chromatography 〈621〉, System Suitability.)

Mode: LC

Detector: Refractive index

Column: 4.6-mm × 15-cm; 5-μm packing L31

Temperatures

Column: 35°

Detector: 35°

Flow rate: 1 mL/min

Injection volume: 100 μL

Run time: NLT 4 times the retention time of zoledronic acid

System suitability

Samples: System suitability solution and Standard solution

Suitability requirements

Resolution: NLT 1.5 between the zoledronic acid and phosphate peaks, System suitability solution

Relative standard deviation: NMT 10.0% for the phosphate and phosphite peaks, Standard solution

Analysis

Samples: Mobile phase, Standard solution, and Sample solution. [Note—Disregard any peak observed in the blank.]

Calculate the percentage of phosphate (determined as phosphoric acid) and phosphite (determined as phosphorus acid) in the portion of Zoledronic Acid taken:

Result = (r_U/r_S) × (C_S/C_U) × 100

r_U = peak response of phosphate or phosphite from the Sample solution

r_S = peak response of phosphate or phosphite from the Standard solution

C_S = concentration of phosphoric acid or phosphorus acid in the Standard solution (mg/mL)

C_U = concentration of Zoledronic Acid in the Sample solution (mg/mL)

Acceptance criteria: See Table 2.

Table 2

^a Determined as phosphoric acid.

^b Determined as phosphorus acid.

5 SPECIFIC TESTS

Bacterial Endotoxins Test 〈85〉: Where the label states that Zoledronic Acid must be subjected to further processing during the preparation of injectable dosage forms, the levels of bacterial endotoxins are such that the requirement under the relevant dosage form monograph(s) in which Zoledronic Acid is used can be met.

Microbial Enumeration Tests 〈61〉 and Tests for Specified Microorganisms 〈62〉: The total aerobic microbial count does not exceed 102 cfu/g, and the total combined molds and yeasts count does not exceed 100 cfu/g.

Water Determination 〈921〉, Method I, Method Ia: For the monohydrate form, 5.0%–7.5%. For the anhydrous form, NMT 0.5%. [Note - Hydranyl solvent and Hydranal titrant 5 are suitable.]

6 ADDITIONAL REQUIREMENTS

Packaging and Storage: Preserve in well-closed containers. Store at room temperature.

Labeling: Where it is intended for use in preparing injectable dosage forms, the label states that it is sterile or must be subjected to further processing during the preparation of injectable dosage forms to ensure acceptable levels of bacterial endotoxins, it is so labeled.

USP Reference Standards 〈11〉

USP Imidazole RS

1H-Imidazole.

C₃H₄N₂68.08

USP Zoledronic Acid RS

USP Zoledronic Acid Related Compound A RS

2-(1H-Imidazol-1-yl)acetic acid.

C₅H₆N₂O₂ 126.12

USP Zoledronic Acid Related Compound B RS

1-Hydroxy-2-[1-(2-hydroxy-2,2-diphosphonoethyl)-1H-imidazol-3-ium-3-yl]-1-phosphonoethylphosphonate.

C₇H₁₆N₂O₁₄P₄ 476.10