Milbemycin Oxime

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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Mixture of milbemycin A₃ oxime and milbemycin A₄ oxime. CAS RN®: 129496-10-2.

Milbemycin A₃ Oxime  C₃₁H₄₃NO₇  541.69

Milbemycin B, 5-demethoxy-28-deoxy-6,28-epoxy-5-(hydroxyamino)-25-methyl-, (6R,25R)-;

(2R,2a′E,2a1′S,4′E,5S,6R,6′R,8′E,11′R,15′S,17a′R,20′Z,20a′R)-2a1′-Hydroxy-20′-(hydroxyimino)-5,6,6′,8′,19′-pentamethyl-2a1′,3,4,5,6,6′,7′,10′,11′,14′,15′,17a′,20′,20a′-tetradecahydro-2′H,17′H-spiro[pyran-2,13′-[11,15]methano[1,5]dioxacyclooctadecino[9,8,7-cd]benzofuran]-17′-one. 

Milbemycin A₄ Oxime

C₃₂H₄₅NO₇ 555.71

Milbemycin B, 5-demethoxy-28-deoxy-6,28-epoxy-25-ethyl-5-(hydroxyamino)-, (6R,25R)-;

(2R,2a′E,2a1′S,4′E,5S,6R,6′R,8′E,11′R,15′S,17a′R,20′Z,20a′R)-6-Ethyl-2a1′-hydroxy-20′-(hydroxyimino)-5,6′,8′,19′-tetramethyl-2a1′,3,4,5,6,6′,7′,10′,11′,14′,15′,17a′,20′,20a′-tetradecahydro-2′H,17′H-spiro[pyran-2,13′-[11,15]methano[1,5]dioxacyclooctadecino[9,8,7-cd]benzofuran]-17′-one. 

1 DEFINITION

Milbemycin Oxime is a mixture of milbemycin A₃ oxime and milbemycin A₄ oxime. It contains NLT 95.0% and NMT 102.0% of the sum of milbemycin A₃ oxime and milbemycin A₄ oxime, calculated on the anhydrous basis. The ratio of milbemycin A₄ oxime is NLT 0.75.

2 IDENTIFICATION

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

B. The retention times of the major peaks of the Sample solution correspond to those of the Standard solution, as obtained in the Assay.

3 ASSAY

3.1 Procedure

Solution A: Water

Solution B: Methanol

Mobile phase: See Table 1.

Table 1

Diluted phosphoric acid solution: 0.3–0.5 mL/L of phosphoric acid in water

Diluent: Acetonitrile and Diluted phosphoric acid solution (75:25)

Standard solution: 0.2 mg/mL of USP Milbemycin Oxime RS in Diluent. Sonicate if necessary to facilitate dissolution.

Sample solution: 0.2 mg/mL of Milbemycin Oxime in Diluent. Sonicate if necessary to facilitate dissolution. This solution must be stored at 5° protected from light.

Chromatographic system

(See Chromatography 〈621〉, System Suitability.)

Mode: LC

Detector: UV 240 nm

Column: 3.0-mm × 10-cm; 3-µm packing L1

Column temperature: 35°

Flow rate: 0.5 mL/min

Injection volume: 10 µL

System suitability

Sample: Standard solution

[Note-The elution order is milbemycin A₃ oxime followed by milbemycin A₄ oxime. (See Table 2 for the relative retention times.)]

Suitability requirements

Resolution: NLT 9 between milbemycin A₃ oxime and milbemycin A₄ oxime

Tailing factor: NMT 2.0 each for milbemycin A₃ oxime and milbemycin A₄ oxime

Relative standard deviation: NMT 0.73% each for milbemycin A₃ oxime and milbemycin A₄ oxime

Analysis

Samples: Standard solution and Sample solution

Calculate the percentage of milbemycin A₃ oxime (C₃₁H₄₃NO₇) in the portion of Milbemycin Oxime taken:

Result = (rᵤ/rₛ) × [(Cₛ × F_A₃)/Cᵤ] × 100

rᵤ = peak response of milbemycin A₃ from the Sample solution

rₛ = peak response of milbemycin A₃ from the Standard solution

Cₛ = concentration of USP Milbemycin Oxime RS in the Standard solution (mg/mL)

F_A₃ = potency of milbemycin A₃ oxime in USP Milbemycin Oxime RS (mg/mg)

Cᵤ = concentration of Milbemycin Oxime in the Sample solution (mg/mL)

Calculate the percentage of milbemycin A₄ oxime (C₃₂H₄₅NO₇) in the portion of Milbemycin Oxime taken:

Result = (rᵤ / rₛ) × [(Cₛ × F_A₄) / Cᵤ] × 100

rᵤ = peak response of milbemycin A₄ from the Sample solution

rₛ = peak response of milbemycin A₄ from the Standard solution

Cₛ = concentration of USP Milbemycin Oxime RS in the Standard solution (mg/mL)

F_A₄ = potency of milbemycin A₄ oxime in USP Milbemycin Oxime RS (mg/mg)

Cᵤ = concentration of Milbemycin Oxime in the Sample solution (mg/mL)

Calculate the percentage of the sum of milbemycin A₃ oxime and milbemycin A₄ oxime in the portion of Milbemycin Oxime taken:

Result = P_A₃ + P_A₄

P_A₃ = percentage of milbemycin A₃ oxime

P_A₄ = percentage of milbemycin A₄ oxime

Calculate the ratio of milbemycin A₃ oxime in the portion of Milbemycin Oxime taken:

Result = P_A₃/(P_A₃+ P_A₄)

P_A₃ = percentage of milbemycin A₃ oxime

P_A₄ = percentage of milbemycin A₄ oxime

Calculate the ratio of milbemycin A₄ oxime in the portion of Milbemycin Oxime taken:

Result = P_A₄/(P_A₃ + P_A₄)

P_A₃ = percentage of milbemycin A₃ oxime

P_A₄ = percentage of milbemycin A₄ oxime

Acceptance criteria

• Sum of milbemycin A₃ oxime and milbemycin A₄ oxime: 95.0%–102.0% on the anhydrous basis
• Ratio of milbemycin A₄ oxime: NLT 0.75

4 IMPURITIES

4.1 Residue on Ignition 〈281〉: NMT 0.5%

4.2 Organic Impurities

Mobile phase, Diluent, Standard solution, Sample solution, Chromatographic system, and System suitability: Proceed as directed in the Assay.

Diluted standard solution: 0.002 mg/mL of USP Milbemycin Oxime RS in Diluent from Standard solution

Analysis

Samples: Sample solution and Diluted standard solution

Calculate the percentage of each impurity in the portion of Milbemycin Oxime taken:

Result = (rᵤ/rₛ) × (Cₛ/Cᵤ) × 100

rᵤ = peak response of each impurity from the Sample solution

rₛ = sum of the peak responses of milbemycin A₃ oxime and milbemycin A₄ oxime from the Diluted standard solution

Cₛ = concentration of USP Milbemycin Oxime RS in the Diluted standard solution (mg/mL)

Cᵤ = concentration of Milbemycin Oxime in the Sample solution (mg/mL)

Acceptance criteria: See Table 2. The reporting level for impurities is 0.10%.

Table 2

ᵃ (1′R,2R,4′S,5S,6R,8′R,10′E,13′R,14′E,16′E,20′R,21′Z,24′S)-6-Ethyl-24′-hydroxy-21′-(hydroxyimino)-5,13′,22′-trimethyl-3,4,5,6-tetrahydrospiro[pyran-2,6′ [3,7,19]trioxatetracyclo[15.6.1.1⁴^,⁸.0²⁰^,²⁴]pentacosa[10,14,16,22]tetraene]-2′-one.

ᵇ (1′R,2R,4′S,5S,6R,8′R,10′E,13′R,14′E,16′E,20′R,21′Z,24′S)-6-Ethyl-20′,24′-dihydroxy-21′-(hydroxyimino)-5,11′,13′,22′-tetramethyl-3,4,5,6-tetrahydrospiro[pyran-2,6′ [3,7,19]trioxatetracyclo[15.6.1.1⁴^,⁸.0²⁰^,²⁴]pentacosa[10,14,16,22]tetraene]-2′-one.

ᶜ (1′R,2R,4′S,5S,6R,8′R,10′E,13′R,14′E,16′E,20′S,24′S)-6-Ethyl-24′-hydroxy-5,11′,13′,22′-tetramethyl-3,4,5,6-tetrahydrospiro[pyran-2,6′-[3,7,19]trioxatetracyclo[15.6.1.1⁴^,⁸.0²⁰^,²⁴]pentacosa[10,14,16,22]tetraene]-2′,21′-dione.

ᵈ (1′R,2R,4′S,5S,6R,8′R,10′E,13′R,14′E,16′E,20′R,21′Z,24′S)-24′-Hydroxy-21′-(hydroxyimino)-6-(propan-2-yl)-5,11′,13′,22′-tetramethyl-3,4,5,6-tetrahydrospiro[pyran-2,6′-[3,7,19]trioxatetracyclo[15.6.1.1⁴^,⁸.0²⁰^,²⁴]pentacosa[10,14,16,22]tetraene]-2′-one.

5 SPECIFIC TESTS

Water Determination 〈921〉, Method I: NMT 3.0%

6 ADDITIONAL REQUIREMENTS

Packaging and Storage: Preserve in tight containers. Store at room temperature, protected from light.

Labeling: Label it to indicate that it is for veterinary use only.

USP Reference Standards 〈11〉

USP Milbemycin Oxime RS