7/11/25, 2:40 PM USP-NF 〈341〉 Antimicrobial Agents—Content
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〈341〉 ANTIMICROBIAL AGENTS—CONTENT
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%.]
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The most commonly used antimicrobial agents include benzyl alcohol; chlorobutanol; phenol; the four homologous esters of p-
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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.
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GENERAL GAS AND LIQUID CHROMATOGRAPHIC METHODS
The general gas chromatography procedures set forth in the following paragraphs are applicable to the quantitative determination of
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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
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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.
• 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 × 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
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Hold Time at
Initial Temperature Temperature Ramp Final Temperature Final Temperature
(°) (°/min) (°) (min)
150 0 150 5
150 10 230 7
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 = (RU/RS) × (CS/CU) × 100
RU
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= peak response ratio of benzyl alcohol to phenol from the Sample solution
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RS = peak response ratio of benzyl alcohol to phenol from the Standard solution
CS = concentration of USP Benzyl Alcohol RS in the Standard solution
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CU = nominal concentration of benzyl alcohol in the Sample solution
• Chlorobutanol
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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
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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 × 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
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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 = (RU/RS) × (CS/CU) × 100
RU = peak response ratio of chlorobutanol to 2,2,2-trichloroethanol from the Sample solution
RS = peak response ratio of chlorobutanol to 2,2,2-trichloroethanol from the Standard solution
CS = concentration of USP Chlorobutanol RS in the Standard solution
CU = nominal concentration of chlorobutanol in the Sample solution
• 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 × 0.32-mm fused-silica; bonded with a 0.5-µm film of phase G16
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Temperatures
Injection port: 200°
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Detector: 310°
Column: See Table 2.
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Table 2
Hold Time at
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Initial Temperature Temperature Ramp Final Temperature Final Temperature
(°) (°/min) (°) (min)
150 0 150 5
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150 10 230 7
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
Samples: Standard solution and Sample solution
Calculate the percentage of the labeled amount of phenol (C6H6O) in the portion of the sample taken:
Result = (RU/RS) × (CS/CU) × 100
RU = peak response ratio of phenol to benzyl alcohol from the Sample solution
RS = peak response ratio of phenol to benzyl alcohol from the Standard solution
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CS = concentration of USP Phenol RS in the Standard solution
CU = nominal concentration of phenol in the Sample solution
• 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 × 3-mm; packing L1
Analytical: 4.6-mm × 15-cm; 5-µm packing L1
Flow rate: 1.3 mL/min
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Injection volume: 10 µL
Run time: 10 min
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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,
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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
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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
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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 = (RU/RS) × (CS/CU) × 100
RU = peak response ratio of methylparaben to ethylparaben from the Sample solution
RS = peak response ratio of methylparaben to ethylparaben from the Standard solution
CS = concentration of USP Methylparaben RS in the Standard solution
CU = 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 = (RU/RS) × (CS/CU) × 100
RU = peak response ratio of propylparaben to ethylparaben from the Sample solution
RS = peak response ratio of propylparaben to ethylparaben from the Standard solution
CS = concentration of USP Propylparaben RS in the Standard solution
CU = 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
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for different product formulations.
• 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 × 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 = (rU/rS) × (CS/CU) × 100
rU = peak response of thimerosal from the Sample solution
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rS = peak response of thimerosal from the Standard solution
CS = concentration of USP Thimerosal RS in the Standard solution
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CU = nominal concentration of thimerosal in the Sample solution
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POLAROGRAPHIC METHOD
• Phenylmercuric Nitrate
Standard stock solution: 0.1 mg/mL of phenylmercuric nitrate in sodium hydroxide solution (1 in 250). Warm, if necessary, to dissolve.
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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
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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[(id)U/(id)S]
C = concentration of phenylmercuric nitrate in the Standard solution (µg/mL)
(id )U = diffusion current of the Sample solution, as the difference between the residual current and the limiting current
(id )S = diffusion current of the Standard solution, as the difference between the residual current and the limiting current
• 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
Auxiliary Information - Please check for your question in the FAQs before contacting USP.
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<341> ANTIMICROBIAL AGENTS--CONTENT Documentary Standards Support SM12020 Small Molecules 1
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DOI: https://doi.org/10.31003/USPNF_M99100_01_01
DOI ref: kjh4o
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