Tetracycline

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

Tetracycline has a potency equivalent to NLT 975 µg/mg of tetracycline hydrochloride (C₂₂H₂₄N₂O₈ · HCl), calculated on the anhydrous basis.

2 IDENTIFICATION

A. SPECTROSCOPIC IDENTIFICATION TESTS 〈197〉 , Ultraviolet-Visible Spectroscopy: 197U Sample solution: 20 µg/mL in 0.25 N sodium hydroxide

Analytical wavelength: 380 nm

Analysis: Measure the absorptivity 6 min after preparation.

Acceptance criteria: Absorptivity, calculated on the anhydrous basis and taking into account the potency of the Reference Standard, is between 104.5% and 111.95% of the absorptivity of USP Tetracy cline Hy drochloride RS.

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

Solution A: Dilute 1 mL of phosphoric acid with water to 1 L.

Solution B: Acetonitrile

Mobile phase: See Table 1.

Table 1

System suitability solution: 25 µg/mL each of USP Anhy drotetracy cline Hy drochloride RS, USP Epitetracy cline Hy drochloride RS, and USP 4- Epianhydrotetracycline Hydrochloride RS, and 100 µg/mL of USP Tetracycline Hydrochloride RS in Solution A

Standard solution: 100 µg/mL of USP Tetracy cline Hy drochloride RS in Solution A

Sample solution: 90 µg/mL of Tetracycline in Solution A Chromatographic system

(See Chromatography 〈621〉, System Suitability.)

Mode: LC

Detector: UV 280 nm

Column: 4.6-mm × 15-cm; 3-µm packing L60. [NOTE—Column with L1 packing is also suitable.] Temperatures

Autosampler: 10°

Column: 50°

Flow rate: 1.0 mL/min

Injection volume: 10 µL

System suitability

Samples: System suitability solution and Standard solution

Suitability requirements

Resolution: NLT 2.5 between epitetracycline and tetracycline; NLT 2.5 between anhydrotetracycline and 4-epianhydrotetracycline, System suitability solution

Tailing factor: NMT 1.5, Standard solution

Relative standard deviation: NMT 0.73%, Standard solution Analysis

Samples: Standard solution and Sample solution

Calculate the potency equivalent, in µg/mg, of tetracycline hydrochloride (C₂₂H₂₄N₂O₈ · HCl) in the portion of Tetracycline taken:

Result = (r_U/r_S) × (C_S/C_U) × P

r_U = peak response of tetracycline from the Sample solution

r_S = peak response of tetracycline from the Standard solution

C_S = concentration of USP Tetracy cline Hy drochloride RS in the Standard solution (µg/mL)

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

P = potency of USP Tetracycline Hydrochloride RS (µg/mg) Acceptance criteria: NLT 975 µg/mg on the anhydrous basis

4 IMPURITIES

Change to read:

ORGANIC IMPURITIES

Solution A, Solution B, Mobile phase, Sample solution, and Chromatographic system: Proceed as directed in the Assay.

System suitability solution: 25 µg/mL each of USP Anhydrotetracycline Hydrochloride RS, USP Epitetracycline Hydrochloride RS, USP 4- Epianhy drotetracy cline Hy drochloride RS, and USP Tetracy cline Hy drochloride RS in Solution A

Standard solution 1: 0.5 µg/mL of USP Anhy drotetracy cline Hy drochloride RS and 3 µg/mL of USP Epitetracy cline Hy drochloride RS in Solution A

Standard solution 2: 2 µg/mL of USP 4-Epianhy drotetracy cline Hy drochloride RS in Solution A

Standard solution 3: 0.1 µg/mL of USP Tetracy cline Hy drochloride RS in Solution A (IRA 1-May-2022)

System suitability

Samples: System suitability solution, Standard solution 1, Standard solution 2, and Standard solution 3 (IRA 1-May-2022)

Suitability requirements

Resolution: NLT 2.5 between epitetracycline and tetracycline; NLT 2.5 between anhydrotetracycline and 4-epianhydrotetracycline, System suitability solution

Relative standard deviation: NMT 2.8% for anhydrotetracycline and epitetracycline, Standard solution 1; NMT 2.8% for 4- epianhydrotetracycline, Standard solution 2; NMT 2.8% for tetracycline, Standard solution 3▲ (IRA 1-May-2022)

Analysis

Samples: Standard solution 1, Standard solution 2, Standard solution 3, (IRA 1-May-2022) and Sample solution

Calculate the percentage of anhydrotetracycline hydrochloride and epitetracycline hydrochloride in the portion of Tetracycline taken:

Result = (r_U/r_S) × (C_S/C_U) × 100

r_U = peak response of anhydrotetracycline or epitetracycline from the Sample solution

r_S = peak response of the corresponding USP Reference Standard from Standard solution 1

C_S = concentration of the corresponding USP Reference Standard in Standard solution 1 (µg/mL)

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

Calculate the percentage of 4-epianhydrotetracycline hydrochloride in the portion of Tetracycline taken:

Result = (r_U/r_S) × (C_S/C_U) × 100

r_U = peak response of 4-epianhydrotetracycline from the Sample solution

r_S = peak response of 4-epianhydrotetracycline from Standard solution 2

C_S = concentration of the USP 4-Epianhy drotetracy cline Hy drochloride RS in Standard solution 2 (µg/mL)

C_U = concentration of Tetracycline in the Sample solution (µg/mL) (IRA 1-May-2022)

Calculate the percentage of 2-acetyl analog or any unspecified impurity in the portion of Tetracycline taken:

Result = (r_U/r_S) × (C_S/C_U) × (M_r1/M_r2) × 100

r_U = peak response of 2-acetyl analog or any unspecified impurity from the Sample solution

r_S = peak response of tetracycline from Standard solution 3 (IRA 1-May-2022)

C_S = concentration of USP Tetracycline Hydrochloride RS in Standard solution 3 (IRA 1-May-2022) (µg/mL)

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

M_r1 = molecular weight of tetracycline, 444.44

M_r2 = molecular weight of tetracycline hydrochloride, 480.90

Acceptance criteria: See Table 2. The reporting threshold is (IRA 1-May-2022) 0.05%.

Table 2

^a 2-Acetyl-2-decarbamoyltetracycline; also known as (4S,4aS,5aS,6S,12aS)-2-Acetyl-4-(dimethylamino)-3,6,10,12,12a-pentahydroxy-6- methyl-4a,5a,6,12a-tetrahydrotetracene-1,11(4H,5H)-dione.

5 SPECIFIC TESTS

OPTICAL ROTATION 〈781S〉 , Procedures, Specific Rotation

Sample solution: 5 mg/mL of tetracycline in 0.1 N hydrochloric acid Acceptance criteria: −260° to −280° on the anhydrous basis

CRYSTALLINITY 〈695〉: Meets the requirements

PH 〈791〉

Sample solution: Prepare in an aqueous suspension (1 in 100). Acceptance criteria: 3.0–7.0

WATER DETERMINATION 〈921〉 , Method I: NMT 13.0%

6 ADDITIONAL REQUIREMENTS

PACKAGING AND STORAGE: Preserve in tight, light-resistant containers.

LABELING: Label it to indicate that it is to be used in the manufacture of nonparenteral drugs only. Change to read:

USP REFERENCE STANDARDS 〈11〉

USP Anhydrotetracycline Hydrochloride RS

(4S,4aS,12aS)-4-(Dimethylamino)-3,10,11,12a-tetrahydroxy-6-methyl-1,12-dioxo-1,4,4a,5,12,12a-hexahydrotetracene-2-carboxamide   monohydrochloride.

C₂₂H₂₂N₂O₇ · HCl    462.88 (IRA 1-May-2022)

USP 4-Epianhydrotetracycline Hydrochloride RS

(4R,4aS,12aS)-4-(Dimethylamino)-3,10,11,12a-tetrahydroxy-6-methyl-1,12-dioxo-1,4,4a,5,12,12a-hexahydrotetracene-2-carboxamide   hydrochloride.

C₂₂H₂₂N₂O₇ · HCl    462.88 (IRA 1-May-2022)

USP Epitetracycline Hydrochloride RS

(4R,4aS,5aS,6S,12aS)-4-(Dimethylamino)-3,6,10,12,12a-pentahydroxy-6-methyl-1,11-dioxo-1,4,4a,5,5a,6,11,12a-octahydrotetracene-2-   carboxamide monohydrochloride.

C₂₂H₂₄N₂O₈ · HCl    480.90 (IRA 1-May-2022)

USP Tetracycline Hydrochloride RS