Tobramycin Inhalation Solution
» Tobramycin Inhalation Solution is a sterile, nonpyrogenic, preservative-free solution of Tobramycin in Water for Injection containing Sodium Chloride. It is prepared with the aid of Sulfuric Acid or Sodium Hydroxide and contains, in each mL, not less than 90.0 percent and not more than 110.0 percent of the labeled amount of tobramycin (C18H37N5O9).
Packaging and storage—
Preserve in low-density, polyethylene, single-use ampules stored in light-resistant foil over-wrapped packaging, in a refrigerator.
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Identification—
The retention time of the major peak in the chromatogram of the Derivatized assay preparation corresponds to that in the chromatogram of the Derivatized standard preparation, as obtained in the Assay.
Absorbance—
The absorbance of the Inhalation Solution determined at 410 nm in a 1-cm cell is not more than 0.24.
Bacterial endotoxins 
85
—
It contains not more than 60 USP Endotoxin Units per mL.
85—
It contains not more than 60 USP Endotoxin Units per mL.
Sterility 71—
It meets the requirements when tested as directed for Membrane Filtration under Test for Sterility of the Product to be Examined.
71—
It meets the requirements when tested as directed for Membrane Filtration under Test for Sterility of the Product to be Examined.
Uniformity of dosage units 905:
meets the requirements.
905:
meets the requirements.
pH 791:
between 5.5 and 6.5.
791:
between 5.5 and 6.5.
Particulate matter 788:
meets the requirements for small-volume injections.
788:
meets the requirements for small-volume injections.
Osmolality and Osmolarity 785—
The osmolality is between 135 and 200 mOsmol per kg.
785—
The osmolality is between 135 and 200 mOsmol per kg.
Related compounds—
Solution A—
Prepare a filtered and degassed mixture of water, acetonitrile, and phosphoric acid (95:5:0.08).
Solution B—
Prepare a filtered and degassed mixture of acetonitrile, water, and phosphoric acid (75:25:0.08).
Mobile phase—
Use variable mixtures of Solution A and Solution B as directed for Chromatographic system. Make adjustments if necessary (see System Suitability under Chromatography 621).
621).
Blank solution—
Use water.
2, 4-Dinitrofluorobenzene reagent and Tris(hydroxymethyl)aminomethane reagent—
Proceed as directed in the Assay.
System suitability stock solution—
Dissolve an accurately weighed quantity of USP Tobramycin RS in water, and adjust with 1 N sulfuric acid to a pH of 6.0. Dilute with water to obtain a solution having a known concentration of about 1.1 mg per mL.
System suitability solution 1—
Dilute the System suitability stock solution quantitatively, and stepwise if necessary, with water to obtain a solution having a known concentration of about 0.22 mg per mL.
System suitability solution 2—
Heat a portion of the System suitability stock solution in a suitable sealed glass container at 100
for 8 to 9 hours. Cool to room temperature, and dilute with water to obtain a solution having a known concentration of about 0.22 mg per mL.
for 8 to 9 hours. Cool to room temperature, and dilute with water to obtain a solution having a known concentration of about 0.22 mg per mL.
Standard solution—
Dilute a portion of Stock standard preparation, prepared as directed in the Assay quantitatively, and stepwise if necessary, with water to obtain a solution having a concentration of 1.10 µg of tobramycin per mL.
Test solution—
Proceed as directed for Assay preparation in the Assay to obtain a solution having a nominal concentration of about 192 µg of tobramycin per mL, based on the label claim.
Derivatization procedure—
[note—Heat all solutions at the same temperature and for the same duration as indicated. Move all flasks to and from the 60 constant-temperature bath at the same time. ] To separate 50-mL flasks transfer 15.0 mL of System suitability solution 1, 15.0 mL of System suitability solution 2, 15.0 mL of Standard solution,15.0 mL of Test solution, and 15.0 mL of Blank solution. To each flask, add 10 mL of 2,4-Dinitrofluorobenzene reagent and 10 mL of Tris(hydroxymethyl)aminomethane reagent, shake, and insert the stopper. Place the flasks in a constant-temperature bath at 60 ± 2, and heat for 50 ± 5 minutes. Remove the flasks from the bath, and allow to stand for 10 minutes. Add acetonitrile to about 2 mL below the 50-mL mark, allow to cool to room temperature, dilute with acetonitrile to volume, and mix. Allow the solutions to stand for 16 hours. The solutions thus obtained are Derivatized system suitability solution 1, Derivatized system suitability solution 2, the Derivatized standard solution, the Derivatized test solution, and the Derivatized blank solution.
constant-temperature bath at the same time. ] To separate 50-mL flasks transfer 15.0 mL of System suitability solution 1, 15.0 mL of System suitability solution 2, 15.0 mL of Standard solution,15.0 mL of Test solution, and 15.0 mL of Blank solution. To each flask, add 10 mL of 2,4-Dinitrofluorobenzene reagent and 10 mL of Tris(hydroxymethyl)aminomethane reagent, shake, and insert the stopper. Place the flasks in a constant-temperature bath at 60 ± 2, and heat for 50 ± 5 minutes. Remove the flasks from the bath, and allow to stand for 10 minutes. Add acetonitrile to about 2 mL below the 50-mL mark, allow to cool to room temperature, dilute with acetonitrile to volume, and mix. Allow the solutions to stand for 16 hours. The solutions thus obtained are Derivatized system suitability solution 1, Derivatized system suitability solution 2, the Derivatized standard solution, the Derivatized test solution, and the Derivatized blank solution.
Chromatographic system (see Chromatography 621)—
The liquid chromatograph is equipped with a 365-nm detector and a 4.6-mm × 25-cm column that contains packing L11. The flow rate is about 1.2 mL per minute. The chromatograph is programmed as follows.
Chromatograph Derivatized system suitability solution 2, and record the peak responses as directed for Procedure: the capacity factor, k¢ determined from tobramycin is not less than 15.5. Chromatograph Derivatized system suitability solution 1, and use the chromatogram to locate the degradation peaks from comparison to Derivatized system suitability solution 2 (deoxystreptamine kanosaminide and nebramine will increase in response in Derivatized system suitability solution 2 when viewed at a 0–10 mAbs unit or 0–5 mV unit full scale). Record the peak responses as directed for Procedure, identifying the tobramycin and the related compound peaks by their relative retention times, which are listed in the table below: the resolution, R, between the nebramine and kanamycin peaks is not less than 1.0; and the relative standard deviation of the peak corresponding to tobramycin for replicate injections of the Derivatized standard solution is not more than 2.0%.
621)—
The liquid chromatograph is equipped with a 365-nm detector and a 4.6-mm × 25-cm column that contains packing L11. The flow rate is about 1.2 mL per minute. The chromatograph is programmed as follows.
| Time (minutes) |
Solution A
(%) |
Solution B
(%) |
 Elution |
|---|---|---|---|
| 0 | 79 | 21 | equilibration |
| 0–14 | 79®66 | 21®34 | linear gradient |
| 14–25 | 66®30 | 34®70 | linear gradient |
| 25–35 | 30 | 70 | isocratic |
| 35–40 | 30®20 | 70®80 | linear gradient |
| 40–50 | 20®5 | 80®95 | linear gradient |
Procedure—
Separately inject equal volumes (about 45 µL) of Derivatized system suitability solution 1, Derivatized system suitability solution 2, the Derivatized standard solution, the Derivatized test solution, and the Derivatized blank solution, record the chromatograms, and measure the peak responses, disregarding any peak corresponding to those obtained from the Derivatized blank solution, and subtracting the quantities of any such peaks found at the relative retention times of 0.36, 0.66, and 0.94 from those found in the Derivatized test solution. For unknown peak determinations, disregard any peaks found in the chromatogram of the Derivatized test solution that correspond to those in the chromatogram of Derivatized system suitability solution 1. Calculate the percentage of each impurity in relation to the tobramycin content of the Inhalation Solution taken by the formula:
(CS / CU)(ri / rS)(100)
in which CS is the concentration, in µg per mL, of USP Tobramycin RS in the Standard solution; CU is the concentration, in µg per mL, of tobramycin in the Test solution; ri is the peak area of any impurity obtained from the Derivatized test solution; and rS is the peak area for tobramycin obtained from the Derivatized standard solution. The specified and unspecified impurities meet the limits shown below.
| Name | Relative Retention Time |
Limit (%) |
|---|---|---|
| Specified unidentifiedimpurity |
0.36 | NMT 0.25% |
| Deoxystreptamine kanosaminide |
0.66 | NMT 0.3% |
| Nebramine | 0.94 | NMT 0.4% |
| Kanamycin B | 0.96 | — |
| Tobramycin | 1.0 | — |
| Any unspecified individualimpurity |
— | NMT 0.1% |
| Total unspecified impurities | — | NMT 0.2% |
| Total impurities | — | NMT 1.0% |
Content of sodium chloride—
Pipet 25 mL of Inhalation Solution into a suitable container. Add between 70 and 100 mL of water. Add 10 mL of an acidic gelatin solution, prepared by dissolving 2 g of gelatin and 50 mL of nitric acid in 1000 mL of water. Titrate potentiometrically with 0.1 N silver nitrate VS using a suitable silver electrode: not less than 90.0% and not more than 110.0% of the labeled amount of sodium chloride is found.
Assay—
Mobile phase—
Dissolve 2.0 g of tris(hydroxymethyl)aminomethane in about 800 mL of water. To this solution add 20 mL of 1 N sulfuric acid, dilute with acetonitrile to obtain 2000 mL of solution, and mix. Allow to cool, and pass through a filter of 0.2-µm or finer porosity. Make adjustments if necessary (see System Suitability under Chromatography 621).
621).
2,4-Dinitrofluorobenzene reagent—
Prepare a solution of 2,4-dinitrofluorobenzene in alcohol having a concentration of 10 mg per mL. [note—This solution may be used for 5 days if refrigerated when not in use. ]
Tris(hydroxymethyl)aminomethane reagent—
Prepare a stock solution of tris(hydroxymethyl)aminomethane in water having a concentration of 15 mg per mL. [note—This stock solution may be used for 1 month if refrigerated when not in use. ] Transfer 40 mL of this stock solution to a 200-mL volumetric flask, add dimethyl sulfoxide with mixing, dilute with dimethyl sulfoxide to volume, and mix. Use this reagent within 4 hours. [note—If kept immersed in an ice-water bath below 10, the reagent may be used for up to 8 hours. ]
, the reagent may be used for up to 8 hours. ]
Stock standard preparation—
Transfer about 55 mg of USP Tobramycin RS, accurately weighed, to a 50-mL volumetric flask, add 1 mL of 1 N sulfuric acid and enough water to dissolve it, dilute with water to volume, and mix.
Standard preparation—
Transfer 10.0 mL of the Stock standard preparation to a second 50-mL volumetric flask, dilute with water to volume, and mix, to obtain a solution having a known concentration of about 0.22 mg of USP Tobramycin RS per mL.
Assay preparation—
Transfer an accurately measured volume of Inhalation Solution to a suitable volumetric flask, and quantitatively dilute with water to obtain a solution having a nominal concentration of about 0.192 mg of tobramycin per mL, based on the label claim.
Derivatization procedure—
[note—Heat all solutions at the same temperature and for the same duration of time as indicated. Move all flasks to and from the 60 constant temperature bath at the same time. ] To separate 50-mL volumetric flasks transfer 4.0 mL of the Standard preparation, 4.0 mL of the Assay preparation, and 4.0 mL of water. To each flask add 10 mL of 2,4-Dinitrofluorobenzene reagent and 10 mL of Tris(hydroxymethyl)aminomethane reagent, shake, and insert the stopper. Place the flasks in a constant temperature bath at 60 ± 2, and heat for 50 ± 5 minutes. Remove the flasks from the bath, and allow to stand for 10 minutes. Add acetonitrile to about 2 mL below the 50-mL mark, allow to cool to room temperature, then dilute with acetonitrile to volume, and mix. The solutions thus obtained are the Derivatized standard preparation, the Derivatized assay preparation, and the Blank preparation, respectively.
constant temperature bath at the same time. ] To separate 50-mL volumetric flasks transfer 4.0 mL of the Standard preparation, 4.0 mL of the Assay preparation, and 4.0 mL of water. To each flask add 10 mL of 2,4-Dinitrofluorobenzene reagent and 10 mL of Tris(hydroxymethyl)aminomethane reagent, shake, and insert the stopper. Place the flasks in a constant temperature bath at 60 ± 2, and heat for 50 ± 5 minutes. Remove the flasks from the bath, and allow to stand for 10 minutes. Add acetonitrile to about 2 mL below the 50-mL mark, allow to cool to room temperature, then dilute with acetonitrile to volume, and mix. The solutions thus obtained are the Derivatized standard preparation, the Derivatized assay preparation, and the Blank preparation, respectively.
Resolution solution—
Prepare a fresh solution of p-naphtholbenzein in acetonitrile having a known concentration of about 0.24 mg per mL. Transfer 2 mL of this solution to a 10-mL volumetric flask, dilute with Derivatized standard preparation to volume, and use promptly.
Chromatographic system (see Chromatography 621)—
The liquid chromatograph is equipped with a 365-nm detector and a 3.9-mm × 30-cm column containing packing L1. The flow rate is about 1.2 mL per minute. Chromatograph the Blank preparation, and record the responses as directed for Procedure. Identify the solvent and reagent peaks. Chromatograph the Resolution solution, and record the responses as directed for Procedure: identify the peaks based on their relative retention times, which are about 0.6 for p-naphtholbenzein and 1.0 for tobramycin; the resolution, R, between the p-naphtholbenzein and tobramycin peaks is not less than 4.0. Chromatograph the Derivatized standard preparation, and record the responses as directed for Procedure: the relative standard deviation for replicate injections of the peak corresponding to tobramycin is not more than 2.0%.
621)—
The liquid chromatograph is equipped with a 365-nm detector and a 3.9-mm × 30-cm column containing packing L1. The flow rate is about 1.2 mL per minute. Chromatograph the Blank preparation, and record the responses as directed for Procedure. Identify the solvent and reagent peaks. Chromatograph the Resolution solution, and record the responses as directed for Procedure: identify the peaks based on their relative retention times, which are about 0.6 for p-naphtholbenzein and 1.0 for tobramycin; the resolution, R, between the p-naphtholbenzein and tobramycin peaks is not less than 4.0. Chromatograph the Derivatized standard preparation, and record the responses as directed for Procedure: the relative standard deviation for replicate injections of the peak corresponding to tobramycin is not more than 2.0%.
Procedure—
Separately inject equal volumes (about 20 µL) of the Derivatized standard preparation and the Derivatized assay preparation into the chromatograph, record the chromatograms, and measure the area responses for the major peaks. Calculate the percent of label claim of tobramycin (C18H37N5O9) in the Inhalation Solution by the formula:
(CS / CU)(P / 1000)(rU / rS)(100)
in which CS is the concentration, in mg per mL, of USP Tobramycin RS in the Standard preparation; CU is the concentration, in mg per mL, of tobramycin in the Assay preparation; (P / 1000) is the potency of tobramycin, converted from µg per mg to mg per mg, in USP Tobramycin RS; and rU and rS are the tobramycin peak area responses obtained from the Derivatized assay preparation and the Derivatized standard preparation, respectively.
Auxiliary Information—
Please check for your question in the FAQs before contacting USP.
| Topic/Question | Contact | Expert Committee |
|---|---|---|
| Monograph | Ahalya Wise, M.S.
Senior Scientific Liaison 1-301-816-8161 |
(SM12010) Monographs - Small Molecules 1 |
| Reference Standards | RS Technical Services 1-301-816-8129 rstech@usp.org |
|
| 85 |
Radhakrishna S Tirumalai, Ph.D.
Principal Scientific Liaison 1-301-816-8339 |
(GCM2010) General Chapters - Microbiology |
| 71 |
Radhakrishna S Tirumalai, Ph.D.
Principal Scientific Liaison 1-301-816-8339 |
(GCM2010) General Chapters - Microbiology |
USP35–NF30 Page 4882
Pharmacopeial Forum: Volume No. 34(2) Page 307