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6.22 CONTENT OF ANTIMICROBIAL AGENTS

      An essential component of injections preserved in multiple-dose containers is the agent or 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 Pharmacopoeial requirement that the presence and amount added of such agent(s) be declared on the label of the container. The methods provided herein for the most commonly used agents are to be used to demonstrate that the declared agent is present but does not exceed the labelled amount by more than 20 per cent of the labelled amount.

      The concentration of an antimicrobial preservative added to a multiple-dose or single-dose parenteral, ear, nose, and eye preparation may diminish during the shelf-life of the product. Because it is recognized that the antimicrobial preservative concentration in a given preparation may decrease during the product’s shelflife, 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 per cent to allow for manufacturing and analytical variations). The quantitative label statement of the preservative content is not intended to mean that the labelled 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 per cent. 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 per ml or 0.1 per cent.)

      The most commonly used agents include the two mercurials, phenylmercuric nitrate and thiomersal and the four homologous esters of p-hydroxybenzoic acid, phenol, benzyl alcohol, and chlorobutanol. The methods for the first two names are polarographic, while quantitative gas chromatography is employed in the determination of the other agents.

GENERAL GAS CHROMATOGRAPHIC METHOD

      The general procedures set forth in the following paragraphs are applicable to the quantitative determination of benzyl alcohol, chlorobutanol, phenol, and the methyl, ethyl, propyl, and butyl esters of phydroxybenzoic acid, the latter being treated as a group, the individual members of which, if present, are capable of separate determination. Prepare the internal standard solution and the standard preparation for each agent as directed individually below. Unless otherwise directed below, prepare the test preparation from accurately measured portions of the internal standard solution and the sample under test, of such size that the concentration of the agent and the composition of the solvent correspond closely to the concentration and composition of the standard preparation.

Benzyl Alcohol

      Carry out determination as described in the “Gas Chromatography” (Appendix 3.4).

      Internal standard solution Dissolve about 380 mg of phenol in 10 ml of methanol contained in a 200-ml volumetric flask. Add water to volume, and mix.

      Standard preparation Dissolve about 180 mg of benzyl alcohol, accurately weighed, in 20.0 ml of methanol contained in a 100-ml volumetric flask. Add internal standard solution to volume, and mix.

      Chromatographic system The chromatographic procedure may be carried out using (a) a glass column (1.8 m × 3 mm), packed with 5 per cent liquid phase of polyethyleneglycol compound (av. mol. wt. about 15,000) (Carbowax 20M or equivalent is suitable) on diatomaceous support (80- to 100- or 100- to 120-mesh), maintained at 140º (Note The diatomaceous support is acid-washed and then water-washed until neutral, but it is not alkali-washed. It may be silanized.), and (b) helium or nitrogen as the carrier gas at a flow rate of 50 ml per minute.

      Procedure Separately inject equal volumes (about 5 μl) of the standard preparation and the test preparation into the chromatograph, and record the chromatograms. Measure the areas under the peaks for benzyl alcohol and phenol of the chromatogram for the standard preparation, designating them P₁ and P₂, respectively. Similarly, determine the corresponding values p₁ and p₂ for the test preparation.

      Calculation Calculate the content, in mg per ml, of benzyl alcohol (C₇H₈O) in the sample taken by the expression:

100(C/V) (p₁/p₂) (P₂/P₁),

in which C is the concentration, in mg per ml, of benzyl alcohol in the standard preparation, and V is the volume, in ml, of the sample under test used in preparing each 100 ml of the test preparation.

Chlorobutanol

      Carry out the determination as described in the “Gas Chromatography” (Appendix 3.4). 

      Internal standard solution Transfer about 140 mg of benzaldehyde to a 100-ml volumetric flask, add 10 ml of methanol, and swirl to dissolve. Dilute with water to volume, and mix.

      Standard preparation Transfer about 125 mg of chlorobutanol, accurately weighed, to a 25-ml volumetric flask. Add 2 ml of methanol, swirl to dissolve, dilute with water to volume, and mix. Transfer 5.0 ml of this solution and 5.0 ml of internal standard solution to a 25-ml flask, and mix to obtain a solution having a known concentration of about 2.5 mg of chlorobutanol per ml.

      Test preparation Dilute, if necessary, an accurately measured volume of the sample under test quantitatively with methanol to obtain a solution containing not more than about 5.0 mg of chlorobutanol per ml. Combine 3.0 ml of this solution with 3.0 ml of internal standard solution, and mix.

      Chromatographic system The chromatographic procedure may be carried out using (a) a glass column (1.8 m × 2 mm), packed with 5 per cent liquid phase of polyethylene glycol compound (av. mol. wt. about 15,000) (Carbowax 20M or equivalent is suitable) on diatomaceous support (80- to 100- or 100- to 120-mesh), maintained at 110º (Note The diatomaceous support is acid-washed and then water-washed until neutral, but it is not alkali-washed. It may be silanized.), (b) helium or nitrogen as the carrier gas at a flow rate of 20 ml per minute, (c) the injection port is maintained at 180º, and (d) the detector is maintained at 220º. Chromatograph the standard preparation, and record the peak responses as directed under Procedure: the resolution factor between the benzaldehyde and the chlorobutanol peak is not less than 2.0, and the relative standard deviation for replicate injections is not more than 2.0 per cent.

      Procedure (Note Use peak areas where peak responses are indicated.) Separately inject equal volumes (about 1 μl) of the standard preparation and the test preparation into the chromatograph, record the chromatograms, and measure the responses of the major peaks. The relative retention times are about 0.8 for benzaldehyde and 1.0 for chlorobutanol.

      Calculation Calculate the quantity, in mg, of chlorobutanol (C₄H₇Cl₃O) in each ml of the sample under test by the expression: 

C(L/D) (R_u/R_s),

in which C is the concentration, in mg per ml, of chlorobutanol calculated on the anhydrous basis, in the standard preparation, L is the labelled quantity, in mg, of chlorobutanol in each ml of the sample under test, D is the concentration, in mg per ml, of chlorobutanol in the test preparation, based on the volume of specimen under test taken and the extent of dilution, and R_u and R_s are the ratios of the chlorobutanol peak to the benzaldehyde peak obtained from the test preparation and the standard preparation, respectively.

Phenol

      Carry out the determination as described in the “Gas Chromatography” (Appendix 3.4).

      Internal standard solution Pipette 1 ml of benzyl alcohol into a 500-ml volumetric flask, add methanol to volume, and mix.

      Standard preparation Dissolve about 75 mg of phenol, accurately weighed, in 7.5 ml of methanol contained in a 100-ml volumetric flask. Add 20.0 ml of internal standard solution, then add water to volume, and mix.

      Chromatographic system The chromatographic procedure may be carried out using (a) a glass column (1.2 m × 3 mm), packed with 5 per cent liquid phase of polyethylene glycol compound (av. mol. wt. about 15,000) (Carbowax 20M or equivalent is suitable) on diatomaceous support (80- to 100- or 100- to 120-mesh), maintained at 145º (Note The diatomaceous support is acid-washed and then water-washed until neutral, but it is not alkali-washed. It may be silanized.), and (b) helium or nitrogen as the carrier gas at a flow rate of 50 ml per minute.

      Procedure Separately inject equal volumes (about 3 μl) of the standard preparation and the test preparation into the chromatograph, and record the chromatograms. Measure the areas under the peaks for phenol and benzyl alcohol of the chromatogram for the standard preparation, designating them P₁ and P₂, respectively. Similarly, measure the corresponding values p₁ and p₂ for the test preparation.

      Calculation Calculate the content, in mg per ml, of phenol (C₆H₆O) in each ml of the sample taken by the expression:

100(C/V) (p₁/p₂) (P₂/P₁),

in which C is the concentration, in mg per ml, of phenol in the standard preparation, and V is the volume, in ml, of the sample under test used in preparing each 100 ml of the test preparation.

Methylparaben and Propylparaben

      Carry out the determination as described in the “Gas Chromatography” (Appendix 3.4).

      Internal standard solution Place about 200 mg of benzophenone in a 250-ml volumetric flask, add ether to volume, and mix.

      Standard preparation Place about 100 mg of methylparaben and 10 mg of propylparaben, each accurately weighed, in a 200-ml volumetric flask, add internal standard solution to volume, and mix. Place 10.0 ml of this solution in a 25-ml conical flask, and proceed as directed under Test preparation, beginning with “Add 3 ml of pyridine”.

      Test preparation Pipette 10 ml of the sample under test and 10 ml of the internal standard solution into a small separator, and transfer the ether layer into a small flask through a funnel containing anhydrous sodium sulfate. Extract the aqueous layer with two 10-ml portions of ether, also filtering the extracts through anhydrous sodium sulfate. Evaporate the combined extracts under a current of dry air until the volume is reduced to about 10 ml, and then transfer the residue to a 25-ml conical flask. Add 3 ml of pyridine, complete the evaporation of the ether, and boil on a hot plate until the volume is reduced to about 1 ml. Cool, and add 1.0 ml of a suitable agent, such as bis(trimethylsilyl) trifluoroacetamide, bis(trimethylsilyl) acetamide, or a mixture of hexamethlyldisilazane and trimethylchlorosilane [2:1 or 3:1 (v/v)]. Mix and allow to stand for not less than 15 minutes.

      Chromatographic system The chromatographic procedure may be carried out using (a) a glass column (1.8 m × 2 mm), packed with 5 per cent liquid phase of dimethylpolysiloxane gum on diatomaceous support (80- to 100- or 100- to 120-mesh), maintained at 150º (Note The diatomaceous support is acid-washed and then waterwashed until neutral, but it is not alkali-washed. It may be silanized.), and (b) helium or nitrogen as the carrier gas at a flow rate of 20 ml per minute.

      Procedure Separately inject equal volumes (about 2 μl) of the silanized solution from the standard preparation and the test preparation into the chromatograph, and record the chromatograms. Measure the areas under the peaks of methylparaben, propylparaben, and benzophenone, designating them P₁, P₂, and P₃ respectively. Similarly, measure the corresponding areas for the silanized solution from the test preparation, designating them p₁, p₂ and p₃, respectively.

      Calculation Calculate the content, in μg per ml, of methylparaben (C₈H₈O₃) in the sample under test by the expression: 

100(C_M/V) (p₁/p₃) (P₃/P₁),

in which C_M is the concentration, in μg per ml, of methylparaben in the standard preparation, and V is the volume, in ml, of the sample taken. Similarly, calculate the content, in μg per ml, of propylparaben (C₁₀H₁₂O₃) in the sample under test by the expression:

100(C_P/V) (p₂/p₃) (P₃/P₂),

in which C_P is the concentration, in μg per ml, of propylparaben in the standard preparation. Ethylparaben and Butylparaben may be determined in a similar manner.

POLAROGRAPHIC METHOD

Phenylmercuric Nitrate

      Standard preparation Dissolve about 100 mg of phenylmercuric nitrate, accurately weighed, in sodium hydroxide solution (1 in 250) contained in a 1000-ml volumetric flask, warming if necessary to effect solution, add the sodium hydroxide solution to volume, and mix. Pipette 10 ml of this solution into a 25-ml volumetric flask, and proceed as directed under Test preparation, beginning with “add 2 ml of a 1 per cent w/v solution of potassium nitrate”.

      Test preparation Pipette 10 ml of the sample under test into a 25-ml volumetric flask, add 2 ml of a 1 per cent w/v solution of potassium nitrate and 10 ml of borate buffer pH 9.2 and adjust to a pH of 9.2 if necessary, by the addition of 2 M nitric acid. Add 1.5 ml of a freshly prepared 0.1 per cent w/v solution of gelatin, then add borate buffer pH 9.2 to volume, and mix. 

      Procedure Pipette a portion of the test preparation into the polarographic cell, and deaerate by bubbling nitrogen through the solution for 15 minutes. Insert the dropping mercury electrode of a suitable polarograph (Appendix 6.25), and record the polarogram from –0.6 to –1.5 volts versus the saturated calomel electrode. Determine the diffusion current of the test preparation, (i_d)_U, as the difference between the residual current and the limiting current. Similarly and concomitantly determine the diffusion current, (i_d)_S, of the standard preparation. Calculate the quantity, in μg, of phenyl mercuric nitrate (C₆H₅HgNO₃.C₆H₅HgOH) in each ml of the sample taken by the expression:

2.5C[(i_d)_U/(i_d)_S],

in which C is the concentration, in μg per ml, of phenylmercuric nitrate in the standard preparation.

Thiomersal

      Standard preparation On the day of use, place about 25 mg of thiomersal, accurately weighed, in a 250-ml volumetric flask, add water to volume, and mix. Protect from light. Pipette 15 ml of this solution into a 25-ml volumetric flask, add 1.5 ml of a freshly prepared 0.1 per cent w/v solution of gelatin, then add a 1 per cent w/v solution of potassium nitrate to volume, and mix.

      Test preparation Pipette 15 ml of the sample under test into a 25-ml volumetric flask, add 1.5 ml of a freshly prepared 0.1 per cent w/v solution of gelatin, add a 1 per cent w/v solution of potassium nitrate to volume, and mix.

      Procedure Transfer a portion of the test preparation to a polarographic cell, and deaerate by bubbling nitrogen through the solution for 15 minutes. Insert the dropping mercury electrode of a suitable polarograph (Appendix 6.25), and record the polarogram from –0.2 to –1.4 volts versus the saturated calomel electrode. Determine the diffusion current, (i_d)_U, as the difference between the residual current and the limiting current. Similarly and concomitantly determine the diffusion current, (i_d)_S, of the standard preparation. Calculate the quantity, in μg of thiomersal (C₉H₉HgO₂S.Na) in each ml of the test specimen taken by the expression:

1.667C[(i_d)_U/(i_d)_S],

in which C is the concentration, in μg per ml, of thiomersal in the standard preparation and the other terms are as defined therein.