Antimicrobial Agents-Content

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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An essential component of Injections preserved in multiple-dose containers is the antimicrobial agent or antimicrobial agents present to reduce the hazard of having introduced, in the course of removing some of the contents, accidental microbial contamination of the contents remaining. It is a Pharmacopeial requirement that the presence and amount added of such antimicrobial agent(s) be declared on the label of the container. This general chapter provides methods for the most commonly used antimicrobial agents. These methods or other suitably validated methods are to be used to demonstrate that the declared antimicrobial agent is present but does not exceed the labeled amount by more than 20%.

The concentration of an antimicrobial preservative added to a multiple-dose or single-dose parenteral, otic, nasal, and ophthalmic preparation may diminish during the shelf life of the product. Therefore, the manufacturer shall determine the lowest level at which the preservative is effective, and the product should be so formulated as to assure that this level is exceeded throughout the product's shelf life. At the time of its manufacture, the product should contain the declared amount of antimicrobial preservative (within ±20% to allow for manufacturing and analytical variations). The quantitative label statement of the preservative content is not intended to mean that the labeled quantity is retained during the shelf life of the product; rather, it is a statement of the amount added, within process limits, and which is not exceeded by more than 20%. An example of such a label statement is "____(unit) added as preservative". [NOTE-"____(unit)" would be a number followed by the unit of measurement, e.g., 0.015 mg/mL or 0.1%.]

The most commonly used antimicrobial agents include benzyl alcohol; chlorobutanol; phenol; the four homologous esters of p-hydroxybenzoic acid (methyl, ethyl, propyl, and butyl parabens); and the two mercurials, phenylmercuric nitrate and thimerosal. The method used for phenylmercuric nitrate is polarographic, whereas quantitative liquid chromatography was used for Thimerosal and the four homologous esters of p-hydroxybenzoic acid. Gas chromatography is used in the determination of phenol, benzyl alcohol, and chlorobutanol.

1 GENERAL GAS AND LIQUID CHROMATOGRAPHIC METHODS

The general gas chromatography procedures set forth in the following paragraphs are applicable to the quantitative determination of benzyl alcohol, chlorobutanol, and phenol. Prepare the Internal standard solution and the Standard solution for each antimicrobial agent as directed below. Unless otherwise directed by the individual monograph, prepare the Sample solution from accurately measured portions of the sample under test and the Internal standard solution such that the concentration of the antimicrobial agent and the composition of the solvent correspond closely to the concentration and composition of the Standard solution. Suggested operating parameters of the gas chromatograph are provided in this section.

The general high-pressure liquid chromatography (HPLC) procedures set forth in the following paragraphs are applicable to the quantitative determination of parabens and Thimerosal. Prepare the Internal standard solution and the Standard solution for each antimicrobial agent as directed below. Unless otherwise directed, prepare the Sample solution from accurately measured portions of the sample under test and the Internal standard solution, if applicable, such that the concentration of the antimicrobial agent and the composition of the solvent is about the same as the concentration and composition of the Standard solution. Suggested operating parameters of the liquid chromatograph are provided in this section.

1.1 BENZYL ALCOHOL

Diluent: Methanol and water (20:80)

Internal standard solution: 3.8 mg/mL of phenol prepared as follows. Dissolve a suitable amount of phenol in 10% of the flask volume of methanol, and dilute with water to volume.

Standard solution: 1.8 mg/mL of USP Benzyl Alcohol RS and 1.5 mg/mL of phenol prepared as follows. Dissolve 180 mg of USP Benzyl Alcohol RS in 20 mL of methanol contained in a 100-mL volumetric flask. Add 40.0 mL of Internal standard solution, and dilute with water to volume.

Chromatographic system

(See Chromatography (621), System Suitability.)

Mode: GC

Detector: Flame ionization

Column: 30-m x 0.32-mm fused-silica; bonded with a 0.5-µm film of phase G16

Temperatures

Injection port: 200°

Detector: 310°

Column: See Table 1.

Table 1

Carrier gas: Helium

Flow rate (constant): 2 mL/min

Injection volume: 1 µL

Split ratio: 10:1

Run time: 20 min

System suitability

Sample: Standard solution

[NOTE-The relative retention times for benzyl alcohol and phenol are about 1.0 and 1.25, respectively.]

Suitability requirements

Resolution: NLT 2.0 between the benzyl alcohol and phenol peaks

Tailing factor: NMT 2.0 for the benzyl alcohol peak

Relative standard deviation: NMT 2.0% for the peak response ratio of benzyl alcohol to phenol

Analysis

Samples: Standard solution and Sample solution

Calculate the percentage of the labeled amount of benzyl alcohol (C₇H₈O) in the portion of the sample taken:

Result = (R_U/R_S) x (C_S/C_U) x 100

R_U = peak response ratio of benzyl alcohol to phenol from the Sample solution

R_S = peak response ratio of benzyl alcohol to phenol from the Standard solution

C_S = concentration of USP Benzyl Alcohol RS in the Standard solution

C_U = nominal concentration of benzyl alcohol in the Sample solution

1.2 CHLOROBUTANOL

Diluent: Methanol and water (50:50)

Internal standard solution: 10 mg/mL of 2,2,2-trichloroethanol in Diluent

Standard stock solution: 5 mg/mL of USP Chlorobutanol RS in methanol

Standard solution: 1.25 mg/mL of USP Chlorobutanol RS and 2 mg/mL of 2,2,2-trichloroethanol prepared as follows. Transfer 2.5 mL of Standard stock solution, 2.0 mL of Internal standard solution, and 0.5 mL of methanol to a 10-mL volumetric flask. Dilute with water to volume.

Sample stock solution: Quantitatively dilute, if necessary, an accurately measured volume corresponding to 2.5 mg/mL of chlorobutanol in water.

Sample solution: Combine 5.0 mL of Sample stock solution with 2.0 mL of Internal standard solution in a 10-mL volumetric flask, and dilute with Diluent to volume.

Chromatographic system

(See Chromatography (621), System Suitability.)

Mode: GC

Detector: Flame ionization

Column: 30-m x 0.32-mm fused-silica; bonded with a 0.25-µm film of phase G16

Temperatures

Injection port: 260°

Detector: 280°

Column: 135°

Carrier gas: Helium

Flow rate: 1 mL/min

Injection volume: 0.5 µL

Split ratio: 10:1

Run time: 12 min

System suitability

Sample: Standard solution

[NOTE-The relative retention times for chlorobutanol and 2,2,2-trichloroethanol are about 1.0 and 1.4, respectively.]

Suitability requirements

Resolution: NLT 2.0 between chlorobutanol and 2,2,2-trichloroethanol

Relative standard deviation: NMT 1.0% for the peak response ratio of chlorobutanol to 2,2,2-trichloroethanol

Analysis

Samples: Standard solution and Sample solution

Calculate the percentage of the labeled amount of chlorobutanol (C₄H₇Cl₃O), on the anhydrous basis, in the portion of the sample taken:

Result = (R_U/R_S) x (C_S/C_U) x 100

R_U = peak response ratio of chlorobutanol to 2,2,2-trichloroethanol from the Sample solution

R_S = peak response ratio of chlorobutanol to 2,2,2-trichloroethanol from the Standard solution

C_S = concentration of USP Chlorobutanol RS in the Standard solution

C_U = nominal concentration of chlorobutanol in the Sample solution

1.3 PHENOL

Internal standard solution: 2 mg/mL of USP Benzyl Alcohol RS in methanol

Standard stock solution: 4 mg/mL of USP Phenol RS in water

Standard solution: 0.4 mg/mL each of USP Phenol RS and USP Benzyl Alcohol RS prepared as follows. Combine 5.0 mL of Standard stock solution with 10.0 mL of Internal standard solution in a 50-mL volumetric flask, and dilute with water to volume.

Chromatographic system

(See Chromatography (621), System Suitability.)

Mode: GC

Detector: Flame ionization

Column: 30-m x 0.32-mm fused-silica; bonded with a 0.5-µm film of phase G16

Temperatures

Injection port: 200°

Detector: 310°

Column: See Table 2.

Table 2

Carrier gas: Helium

Flow rate (constant flow): 2 mL /min

Injection volume: 1 µL

Split ratio: 10:1

Run time: 20 min

System suitability

Sample: Standard solution

[NOTE-The relative retention times for benzyl alcohol and phenol are about 0.85 and 1.0, respectively.]

Suitability requirements

Resolution: NLT 2.0 between benzyl alcohol and phenol

Tailing factor: NMT 2.0 for the phenol peak

Relative standard deviation: NMT 1.0% for the peak response ratio of phenol to benzyl alcohol

Analysis

Calculate the percentage of the labeled amount of phenol (C₆H₆O) in the portion of the sample taken:

Samples: Standard solution and Sample solution

Result = (R_U/R_S) x (C_S/C_U) x 100

R_U = peak response ratio of phenol to benzyl alcohol from the Sample solution

R_S = peak response ratio of phenol to benzyl alcohol from the Standard solution

C_S = concentration of USP Phenol RS in the Standard solution

C_U = nominal concentration of phenol in the Sample solution

1.4 METHYLPARABEN AND PROPYLPARABEN

Buffer: 7 g/L of monobasic potassium phosphate in water

Mobile phase: Methanol and Buffer (65:35)

Internal standard solution: 0.013 mg/mL of USP Ethylparaben RS in Mobile phase

System suitability solution: 0.01 mg/mL each of USP Butylparaben RS, USP Propylparaben RS, USP Ethylparaben RS, USP Methylparaben RS, and p-hydroxybenzoic acid in Mobile phase

Standard stock solution: 0.2 mg/mL of USP Methylparaben RS and 0.03 mg/mL of USP Propylparaben RS in Mobile phase

Standard solution: Combine 5 mL of Standard stock solution with 5 mL of Internal standard solution, and extract three times with 10-mL aliquots of diethyl ether. Filter the combined ether layers through anhydrous sodium sulfate. Evaporate the ether extract to dryness, and dissolve the residue in 50 mL of Mobile phase.

Sample solution: Combine 5 mL of the specimen under test with 5 mL of Internal standard solution, and extract three times with 10-mL aliquots of diethyl ether. Filter the combined ether layers through anhydrous sodium sulfate. Evaporate the ether extract to dryness, and dissolve the residue in 50 mL of Mobile phase.

Chromatographic system

(See Chromatography (621), System Suitability.)

Mode: LC

Detector: UV 272 nm

Columns

Guard: 4.0-mm x 3-mm; packing L1

Analytical: 4.6-mm x 15-cm; 5-µm packing L1

Flow rate: 1.3 mL/min

Injection volume: 10 µL

Run time: 10 min

System suitability

Samples: System suitability solution and Standard solution

[NOTE-The relative retention times for p-hydroxybenzoic acid, methylparaben, ethylparaben, and propylparaben are about 0.58, 1.0, 1.4, and 2.1, respectively.]

Suitability requirements

Resolution: NLT 2.0 between p-hydroxybenzoic acid and methylparaben, NLT 2.0 between methylparaben and ethylparaben; System suitability solution

Tailing factor: NMT 2.0 for the methylparaben and propylparaben peaks, Standard solution

Relative standard deviation: NMT 2.0% for the peak response ratio of methylparaben to ethylparaben, NMT 2.0% for the peak response ratio of propylparaben to ethylparaben; System suitability solution

Analysis

Samples: Standard solution and Sample solution

Calculate the percentage of the labeled amount of methylparaben (C₈H₈O₃) in the portion of the sample taken:

Result = (R_U/R_S) x (C_S/C_U) x 100

R_U = peak response ratio of methylparaben to ethylparaben from the Sample solution

R_S = peak response ratio of methylparaben to ethylparaben from the Standard solution

C_S = concentration of USP Methylparaben RS in the Standard solution

C_U = nominal concentration of methylparaben in the Sample solution

Calculate the percentage of the labeled amount of propylparaben (C₁₀H₁₂O₃) in the portion of the sample taken:

Result = (R_U/R_S) x (C_S/C_U) x 100

R_U = peak response ratio of propylparaben to ethylparaben from the Sample solution

R_S = peak response ratio of propylparaben to ethylparaben from the Standard solution

C_S = concentration of USP Propylparaben RS in the Standard solution

C_U = nominal concentration of propylparaben in the Sample solution

Ethylparaben and butylparaben may be determined in a similar manner using appropriate internal standard solutions. However, because the extraction recovery is matrix dependent, the user should verify the suitability of the procedure for their drug product and for different product formulations.

1.5 THIMEROSAL

Solution A: Trifluoroacetic acid and water (0.5:1000)

Mobile phase: Methanol and Solution A (60:40)

Standard solution: 25 µg/mL of USP Thimerosal RS in water

Chromatographic system

(See Chromatography (621), System Suitability.)

Mode: LC

Detector: UV 222 nm

Column: 2.1-mm x 10-cm; 2-µm packing L1

Autosampler temperature: 4°

Flow rate: 0.35 mL/min

Injection volume: 2.5 µL

System suitability

Sample: Standard solution

Tailing factor: NMT 1.5

Relative standard deviation: NMT 1.0%

Analysis

Samples: Standard solution and Sample solution

Calculate the percentage of the labeled amount of thimerosal (C₉H₉HgNaO₂S) in the portion of the sample taken:

Result = (r_U/r_S) x (C_S/C_U) x 100

r_U = peak response of thimerosal from the Sample solution

r_S = peak response of thimerosal from the Standard solution

C_S = concentration of USP Thimerosal RS in the Standard solution

C_U = nominal concentration of thimerosal in the Sample solution

2 POLAROGRAPHIC METHOD

2.1 PHENYLMERCURIC NITRATE

Standard stock solution: 0.1 mg/mL of phenylmercuric nitrate in sodium hydroxide solution (1 in 250). Warm, if necessary, to dissolve.

Standard solution: Pipet 10 mL of Standard stock solution into a 25-mL volumetric flask, and proceed as directed under Sample solution beginning with "add 2 mL of potassium nitrate solution (1 in 100)".

Sample solution: Pipet 10 mL of the specimen under test into a 25-mL volumetric flask, add 2 mL of potassium nitrate solution (1 in 100) and 10 mL of pH 9.2 alkaline borate buffer (see in Buffer Solutions in the section Reagents, Indicators, and Solutions), and adjust to a pH of 9.2, if necessary, by the addition of 2 N nitric acid. Add 1.5 mL of freshly prepared gelatin solution (1 in 1000), then add the pH 9.2 alkaline borate buffer to volume.

Analysis: Pipet a portion of the Sample solution into the polarographic cell, and deaerate by bubbling nitrogen through the solution for 15 min. Insert the dropping mercury electrode of a suitable polarograph (see Polarography (801)), and record the polarogram from -0.6 to -1.5 volts versus the saturated calomel electrode.

Calculate the quantity, in µg/mL, of phenylmercuric nitrate (C₆H₅HgNO₃) in the portion of the sample taken:

Result = 2.5C[(i_d)_U/(i_d)_S]

C = concentration of phenylmercuric nitrate in the Standard solution (µg/mL)

(i_d)_U = diffusion current of the Sample solution, as the difference between the residual current and the limiting current

(i_d)_S = diffusion current of the Standard solution, as the difference between the residual current and the limiting current

2.2 USP REFERENCE STANDARDS (11)

USP Benzyl Alcohol RS

USP Butylparaben RS

USP Chlorobutanol RS

USP Ethylparaben RS

USP Methylparaben RS

USP Phenol RS

USP Propylparaben RS

USP Thimerosal RS