4-AMINOPHENOL IN ACETAMINOPHEN-CONTAINING DRUG PRODUCTS

This article is compiled based on the United States Pharmacopeia (USP) – 2025 Edition

Issued and maintained by the United States Pharmacopeial Convention (USP)

DOWNLOAD PDF HERE

1 INTRODUCTION

This chapter provides a procedure and acceptance criterion (limit) to control the principal degradation product of acetaminophen, 4-aminophenol, an impurity that can form by hydrolysis of acetaminophen.

2 SOLUTION PREPARATIONS

All solution preparations that contain Acetaminophen or 4-aminophenol should be protected from light and should be stored only for as long as can be supported by solution stability data acquired during verification under actual conditions of use.

Buffer: 4.0 g/L of sodium citrate dihydrate and 1.5 g/L of anhydrous citric acid, in water

Diluent: Acetonitrile and Buffer (10:90)

Solution A: 10 mM phosphate buffer prepared as follows. Add 0.60 g of monobasic potassium phosphate and 0.82 g of anhydrous dibasic sodium phosphate to a 1-L volumetric flask. Dissolve and dilute with water to volume to a pH of 7.0.

Solution B: Water

Solution C: Acetonitrile

Mobile phase: See Table 1.

Table 1

Standard stock solution: 25 µg/mL of USP 4-Aminophenol RS in Diluent. Prepare fresh in conjunction with the other solution preparations described below. Discard after 4 h or as supported by solution stability data. [Note-See Chromatographic Adjustments, solution stability, below.]

System suitability solution: 2.5 µg/mL of USP 4-Aminophenol RS in Diluent, from the Standard stock solution

Sample stock solution: Nominally 10 mg/mL of acetaminophen from a suitable quantity of drug product in Diluent. [Note-Either component of the Diluent may be introduced to the drug product first, followed by addition of the other component to maintain the proportions of acetonitrile and Buffer and to achieve the appropriate final volume defined for the Diluent.]

[Note-It is recommended that the Sample solution and Standard solution be prepared concurrently within a narrow window of time (e.g., 30 min) for each drug product sample.]

Standard solution: Add 25.0 mL of the Sample stock solution and 15.0 mL of the Standard stock solution to a 50-mL volumetric flask, and dilute with Diluent to volume. Pass through a suitable filter of 0.45-µm pore size, discarding the first 3 mL of filtrate.

Sample solution: Add 25.0 mL of the Sample stock solution to a 50-mL volumetric flask, and dilute with Diluent to volume. Pass through a suitable filter of 0.45-µm pore size, discarding the first 3 mL of filtrate.

3 CHROMATOGRAPHIC METHOD

Chromatographic system

• (See Chromatography 〈621〉, System Suitability.)
• Mode: LC
• Detector: UV 300 nm
• Column: 4.6-mm × 15-cm; 5-µm packing L85
• Column temperature: 30°
• Flow rate: 1 mL/min
• Injection volume: 10 µL

System suitability

• Samples: System suitability solution and Standard solution
• [Note-The typical retention time for 4-aminophenol is about 4.2–5.3 min.]
• Suitability requirements
• Resolution: NLT 1.0 between 4-aminophenol and the nearest peak, Standard solution
• Tailing factor: NMT 1.5 for the 4-aminophenol peak, Standard solution
• Relative standard deviation: NMT 5.0%, Standard solution
• Signal-to-noise ratio: NLT 20 for the 4-aminophenol peak, System suitability solution

4 ANALYSIS

Samples: Standard solution and Sample solution

Inject the Sample solution and Standard solution for each drug product sample sequentially, i.e., back-to-back.

Calculate the percentage of 4-aminophenol (C₆H₇NO) relative to acetaminophen in the portion of drug product taken:

Result = [r_u/(r_s − r_u)] × (W_s/W_u) × 100

r_u = peak response of 4-aminophenol from the Sample solution

r_s = peak response of 4-aminophenol from the Standard solution

W_s = amount of USP 4-Aminophenol RS added to the Standard solution (mg)

W_u = amount of acetaminophen in the Sample solution (mg)

Acceptance criteria (unless otherwise stated in the monograph): NMT 0.15% of 4-aminophenol relative to acetaminophen

5 CHROMATOGRAPHIC ADJUSTMENTS

The retention time of 4-aminophenol can be tuned to achieve specificity for a given product matrix. This allowance supersedes provisions in 〈621〉 for adjusting chromatographic conditions and is intended to provide a measure of flexibility when needed.

Suggestions for changing 4-aminophenol retention are given in Table 2. The use of a ternary mobile phase system affords ready changes to the ionic strength (water from Solution B) and organic strength (acetonitrile from Solution C), but this can be simplified to a binary mobile phase system.

Table 2

Adjustments to the chromatographic procedure may require verification or validation. See Validation of Compendial Procedures 〈1225〉 and Verification of Compendial Procedures 〈1226〉 for guidance. Adjusted chromatographic conditions must meet all system suitability requirements.

Solution stability must be verified under actual conditions of use to ensure 4-aminophenol is stable in the Sample solution and the Standard solution as evidenced by NMT ±10% change of the 4-aminophenol peak areas.

6 USP Reference Standards 〈11〉

USP 4-Aminophenol RS

4-Amino-1-hydroxybenzene.

C₆H₇NO 109.13