Doxycycline Hyclate

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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2-Naphthacenecarboxamide, 4-(dimethylamino)-1,4,4a,5,5a,6,11,12a-octahydro-3,5,10,12,12a-pentahydroxy-6-methyl-1,11-dioxo-,monohydrochloride, compound with Ethanol (2:1), monohydrate, [4S-(4α,4aα,5α,5aα,6α,12aα)]-;

(4S,4aR,5S,5aR,6R,12aS)-4-(Dimethylamino)-1,4,4a,5,5a,6,11,12a-octahydro-3,5,10,12,12a-pentahydroxy-6-methyl-1,11-dioxo-2-naphthacenecarboxamide monohydrochloride, compound with ethyl alcohol (2:1), monohydrate CAS RN^®: 24390-14-5; UNII: 19XTS3T51U

1 DEFINITION

Doxycycline Hyclate has a potency equivalent to NLT 800 µg/mg and NMT 920 µg/mg of doxycycline (C₂₂H₂₄N₂O₈).

2 IDENTIFICATION

A. SPECTROSCOPIC IDENTIFICATION TESTS (197), Infrared Spectroscopy: 197A or 197K

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

C. IDENTIFICATION TESTS GENERAL GENERAL (191), Chemical Identification Tests, Chloride: Meets the requirements

3 ASSAY

PROCEDURE

Protect solutions containing doxycycline from light.

Solution A: Transfer 3.1 g of monobasic potassium phosphate and 0.5 g of edetate disodium to a 1000-mL volumetric flask. Add about 850 mL of water and 0.5 mL of triethylamine, and mix. Dilute with water to volume and adjust with 1 N sodium hydroxide to a pH of 8.5 ± 0.1.

Solution B: Methanol

Mobile phase: See Table 1.

Diluent: 0.01 N hydrochloric acid

Standard solution: 0.1 mg/mL of USP Doxycycline Hyclate RS in Diluent. Sonicate as needed to dissolve.

Sample solution: 0.1 mg/mL of Doxycycline Hyclate in Diluent. Sonicate as needed to dissolve.

Chromatographic system

(See Chromatography (621), System Suitability.)

Mode: LC

USP-NF Doxycycline Hyclate

Detector: UV 270 nm

Column: 2.1-mm x 5-cm; 1.7-um packing 17

Column temperature: 60"

Flow rate: 0.6 mL/min

Injection volume: 5 µL

System suitability

Sample: Standard solution

Suitability requirements

Tailing factor: NMT 1.5

Relative standard deviation: NMT 1.0%

Analysis

Samples: Standard solution and Sample solution

Calculate the quantity of doxycycline (C₂₂H₂₄N₂O₈), in µg/mg, in the portion of Doxycycline Hyclate taken:

Result = (r_U/(r_S) × (C_S /C_U) × P 

r_U = peak response from the Sample solution

r_S = peak response from the Standard solution

C_S = concentration of USP Doxycycline Hyclate RS, in the Stardard solution (mg/ml)

C_U = nominal concentration of doxycycline in the Sample sulation (mg/ml)

P = potency of doxycycline in USP Doxycycline Hyclate RS (µg/mg)

Acceptance criteria: 800-920 µg/mg

4 IMPURITIES

Change to read:

ORGANIC IMPURITIES

AL

Mobile phase, Diluent, and Chromatographic system: Proceed as directed in the Assay.

System suitability stock solution 1: 1 mg/ml each of USP Methacycline Hydrochloride RS and USP Doxycycline Related Compound A RS in Diluent

System suitability stock solution 2: 1.2 mg/ml of USP Doxycycline Hvclate RS in Diluent

System suitability solution: Transfer 5 ml. of System suitability stock solution 2 to a 25-ml, volumetric flask, heat on a steam bath for 60 min, and evaporate to dryness on a hot plate, taking care not to char the residue. Dissolve the residue in Diluent, add 0.5 mL of System suitability stock solution 1, and dilute with Diluent to volume. Pass the solution through a suitable filter and use the filtrate. This solution contains a mixture of 4-epidoxycycline, methacycline, doxycycline related compound A, and doxycycline. When stored in a refrigerator, this solution may be used for 14 days

Sensitivity solution: 0.001 mg/ml of USP Doxycycline Hyclate RS in Diluent

Standard solution: 0.002 mg/ml each of USP Doxycycline Hyclate RS and USP Methacycline Hydrochloride RS in Diluent

Sample solution: 2 mg/mL of Doxycycline Hyclate in Diluent. Sonicate as needed to dissolve

System suitability

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

Suitability requirements

Resolution: NLT 1.5 between methacycline and 4-epidoxycycline; NLT 1.5 between 4-epidoxycycline and doxycycline related compound A NLT 2.0 between doxycycline related compound A and doxycycline, System suitability solution

Relative standard deviation: NMT 5.0% each for the doxycycline and methacycline peaks, Standard solution

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

Analysis

Samples: Standard solution and Sample solution

Calculate the percentage of methacycline in the portion of Doxycycline Hyclate taken:

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

r_U = peak response of methacycline from the Sample solution

r_S = peak response of methacycline from the Standard solution

C_S = concentration of USP Doxycycline Hyclate RS, in the Stardard solution (mg/ml)

C_U = concentration ofof Doxycycline Hyclate in the Sample sulation (mg/ml)

P = potency of methacycline in USP Methacycline Hydrochloride RS (μg/mL)

F = conversion factor 0.001 mg/μg

Calculate the percentage of 4-epidoxycycline, doxycycline related compound A, doxycycline related compound F, and any individual unspecified impurity in the portion of Doxycycline Hyclate taken.

Result = (r_U/(r_S) × (C_S /C_U) × P x F₁/F₂ x 100

r_U = peak response of methacycline from the Sample solution

r_S = peak response of methacycline from the Standard solution

C_S = concentration of USP Doxycycline Hyclate RS, in the Stardard solution (mg/ml)

C_U = concentration ofof Doxycycline Hyclate in the Sample sulation (mg/ml)

P = potency of methacycline in USP Methacycline Hydrochloride RS (μg/mL)

F₁ = conversion factor 0.001 mg/μg

F₂ = relative response factor (see Table 2)

Acceptance criteria: See Jabin 2. The reporting threshold is 0.05%

^a(4R,4aR,5S,5aR,6R,12aS)-4-(Dimethylamino)-1,4,4a,5,5a,6,11,12a-octahydro-3,5,10,12,12a-pentahydroxy-6-methyl-1,11-dioxo-2-
naphthacenecarboxamide. Main degradation product.

^b (4S,4aR,5S,5aR,6R,12aS)-2-Acetyl-4-(dimethylamino)-3,5,10,12,12a-pentahydroxy-6-methyl-4a,5a,6,12a-tetrahydrotetracene-1,11(4H,5H)-
dione.

CONTENT OF ETHANOL

Diluent: Dimethylacetamide

Standard solution: 2.5 mg/ml of ethanol in Diluent

Sample solution: 50mg / m * L Doxycycline Hyclate in Diluent prepared as follows. Transfer 100 mg of Doxycycline Hyclate to a 10-ml flasik

fitted with a septum, add 2 mL of Diluent, and vortex to dissolve

(See Chromatography (621) System Suitability)

Mode: GC

Chromatographic system

Detector: Flame ionization

Columns

Guard: 1-m deactivated silica

Analytical: 0.32mm * 30m : fused silica, coated with a 1.0 µm film of phase 625

Temperatures

Injection port: 150°

Detector: 250°

Column: See Table 3

Carrier gas: Helium

Flow rate: 5 mL/min

Injection volume: 1 L

Injection type: Split, split ratio 6:1

Run time: NLT 2.2 times the retention time of ethanol

System suitability

Sample: Standard solution

Suitability requirements

Tailing factor: NMT 2.2

Relative standard deviation: NMT 10%

Analysis

Samples: Standard solution and Sample solution.

Calculate the percentage of ethanol in the portion of Doxycycline Hyclate taken

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

r_U = peak response of ethanol from the Sample solution

r_S = peak response of ethanol  from the Standard solution

C_S = concentration of ethanol in the Stardard solution (mg/ml)

C_U = concentration of Doxycycline Hyclate in the Sample sulation (mg/ml)

Acceptance criteria: 4.3%–5.5%; for veterinary products, 4.3%–6.0%

5 SPECIFIC TESTS

CRYSTALLINITY (695): Meets the requirements.

PH(791)

Sample solution: Nominally 10 mg/mL of doxycycline from Doxycycline Hyclate

Acceptance criteria: 2.0-3.0

WATER DETERMINATION (921), Method: 1.4%-2.8%

STERILITY THATS (71); Where the label states that Doxycycline Hyclate is sterile, it meets the requirements. If the membrane filtration test is

used, use Fluid D instead of Fluid A

BACTERIAL ENDOTOKONS TEST (85): Where the label states that Doxycycline Hyclate is sterile or must be subjected to further processing during the preparation of injectable dosage forms, it has NMT 1.14 USP Endotoxin Units/mg of doxycycline.

ADDITIONAL REQUIREMENTS

PACKAGING AND STORAGE: Preserve in tight containers, protected from light.

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.