Bupivacaine Hydrochloride and Epinephrine Injection

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

Bupivacaine Hydrochloride and Epinephrine Injection is a sterile solution of Bupivacaine Hydrochloride and Epinephrine or Epinephrine Bitartrate in Water for Injection. It contains NLT 93.0% and NMT 107.0% of the labeled amount of bupivacaine hydrochloride (C₁₈H₂₈N₂O·HCl). The content of epinephrine (C₉H₁₃NO₃) does not exceed 0.001% (1 in 100,000). It contains the equivalent of NLT 90.0% and NMT 115.0% of the labeled amount of epinephrine (C₉H₁₃NO₃).

2 IDENTIFICATION

A.

Procedure 1

Sample solution: Nominally 2 mg/mL of bupivacaine hydrochloride in 0.01 N hydrochloric acid from Injection

Analysis: Proceed as directed in Identification—Organic Nitrogenous Bases 〈181〉, beginning with “Transfer the liquid to a separator”.

Acceptance criteria: Meets the requirements

Procedure 2

Sample solution: Use the Sample solution from Procedure 1: Bupivacaine Hydrochloride in the Assay.

Acceptance criteria: The retention time of the bupivacaine peak of the Sample solution corresponds to that of the Standard solution, as obtained in Procedure 1: Bupivacaine Hydrochloride in the Assay.

B.

Sample: Nominally equivalent to 50 µg of epinephrine from Injection

Analysis: Pipet the Sample into a suitable container, add 0.1 mL of Ferro-citrate solution and 2.0 mL of Buffer solution (prepared as directed in Epinephrine Assay 〈391〉), mix, and allow the solution to stand for 10 min. Filter the solution.

Acceptance criteria: The filtrate is violet in color and may turn brownish.

3 ASSAY

Procedure 1: Bupivacaine Hydrochloride

Buffer: 1.94 g/L of monobasic potassium phosphate and 2.48 g/L of dibasic potassium phosphate in water. Adjust, if necessary, with 1 N potassium hydroxide or 1 M phosphoric acid to a pH of 6.8.

Mobile phase: Acetonitrile and Buffer (65:35). Adjust, if necessary, with 1 M phosphoric acid to a pH of 7.7 ± 0.2. Pass the solution through a membrane filter of 1-µm or finer pore size, and degas.

Internal standard solution: 1.3 mg/mL of dibutyl phthalate in methanol

Standard solution: 0.5 mg/mL of USP Bupivacaine Hydrochloride RS, prepared as follows. In a 100-mL volumetric flask, dissolve 50 mg of USP Bupivacaine Hydrochloride RS in 10.0 mL of water, using sonication if necessary. Add 10 mL of Internal standard solution, and dilute with methanol to volume.

Sample solution: Nominally 0.5 mg/mL of bupivacaine hydrochloride, prepared as follows. In a 100-mL volumetric flask, transfer an amount of Injection equivalent to 50 mg of bupivacaine hydrochloride, add 10.0 mL of Internal standard solution, and dilute with methanol to volume.

Chromatographic system

(See Chromatography 〈621〉, System Suitability.)

Mode: LC

Detector: UV 263 nm

Column: 4-mm × 30-cm; packing L1

Flow rate: 2 mL/min

Injection volume: 20 µL

System suitability

Sample: Standard solution

[Note—The relative retention times for bupivacaine and dibutyl phthalate are about 1.0 and 1.2, respectively.]

Suitability requirements

Resolution: NLT 2.0 between bupivacaine and dibutyl phthalate

Relative standard deviation: NMT 1.0% for the ratio of bupivacaine to the internal standard from three replicate Injections

Analysis

Samples: Standard solution and Sample solution

Calculate the percentage of the labeled amount of bupivacaine hydrochloride (C₁₈H₂₈N₂O·HCl) in the portion of Injection taken:

Result = (R₁ / R₂) × (C₁ / C₂) × 100

Where:

R₁ = peak response ratio of bupivacaine to the internal standard from the Sample solution

R₂ = peak response ratio of bupivacaine to the internal standard from the Standard solution

C₁ = concentration of USP Bupivacaine Hydrochloride RS, calculated on the anhydrous basis, in the Standard solution (mg/mL)

C₂ = nominal concentration of bupivacaine hydrochloride in the Sample solution (mg/mL)

Acceptance criteria: 93.0%–107.0%

Procedure 2: Epinephrine

Mobile phase: Prepare a mixture of water, methanol, and 2 M monobasic sodium phosphate (900:50:50), containing 40 mg/L of edetate disodium, 0.4 mL/L of phosphoric acid, and 0.4 g/L of sodium 1-octanesulfonate. Make adjustments, if necessary, to obtain a retention time of NLT 11 min for the epinephrine peak.

System suitability solution: 2 µg/mL each of epinephrine bitartrate and dopamine hydrochloride in Mobile phase

Standard solution: 2 µg/mL of USP Epinephrine Bitartrate RS in Mobile phase

Sample solution: Nominally 1 µg/mL of epinephrine, prepared as follows. In a 25-mL volumetric flask, transfer an amount of Injection equivalent to 25 µg of epinephrine, and dilute with Mobile phase to volume.

Chromatographic system

(See Chromatography 〈621〉, System Suitability.)

Mode: LC

Detector: Electrochemical held at a potential of +0.75 V

Column: 4.6-mm × 25-cm; packing L1

Flow rate: 1.2 mL/min

Injection volume: 20 µL

System suitability

Samples: System suitability solution and Standard solution

[Note—The relative retention times for epinephrine and dopamine are about 1.0 and 2, respectively.]

Suitability requirements

Resolution: NLT 6.0 between the epinephrine and dopamine peaks, System suitability solution

Relative standard deviation: NMT 2.0%, Standard solution

Analysis

Samples: Standard solution and Sample solution

Calculate the percentage of the labeled amount of epinephrine (C₉H₁₃NO₃) in the portion of Injection taken:

Result = (r₁ / r₂) × (C₁ / C₂) × (M₁ / M₂) × 100

Where:

r₁ = peak response of epinephrine from the Sample solution

r₂ = peak response of epinephrine from the Standard solution

C₁ = concentration of USP Epinephrine Bitartrate RS in the Standard solution (µg/mL)

C₂ = nominal concentration of epinephrine in the Sample solution (µg/mL)

M₁ = molecular weight of epinephrine, 183.21

M₂ = molecular weight of epinephrine bitartrate, 333.30

Acceptance criteria: 90.0%–115.0%

4 SPECIFIC TESTS

Color and Clarity

Standard solution: Transfer 2.0 mL of 0.100 N iodine VS to a 500-mL volumetric flask and dilute with water to volume.

Sample solution: Injection

Analysis 1: Visually examine a portion of the Sample solution in a suitable clear glass test tube against a white background.

Acceptance criteria 1: The Sample solution is not pinkish, and it contains no precipitate.

Analysis 2: Perform Analysis 2 if any yellow color is observed in the Sample solution. Concomitantly determine the absorbances of the Sample solution and the Standard solution in 1-cm cells with a suitable spectrophotometer set at 460 nm.

Acceptance criteria 2: The absorbance of the Sample solution does not exceed that of the Standard solution.

Bacterial Endotoxins Test 〈85〉: NMT 1.6 USP Endotoxin Units/mg of bupivacaine hydrochloride

 pH 〈791〉: 3.3–5.5

Other Requirements: It meets the requirements in Injections and Implanted Drug Products 〈1〉.

5 ADDITIONAL REQUIREMENTS

Packaging and Storage: Preserve in single-dose or multiple-dose containers, preferably of Type I glass, protected from light. Injection labeled to contain 0.5% or less of bupivacaine hydrochloride may be packaged in 50-mL multiple-dose containers.

Labeling: The label indicates that Injection is not to be used if its color is pinkish or darker than slightly yellow, or if it contains a precipitate.

USP Reference Standards 〈11〉

USP Bupivacaine Hydrochloride RS

USP Epinephrine Bitartrate RS