EUROPEAN PHARMACOPOEIA 7.

0 Tobramycin

suitable for the purpose, but other methods can also be used. Drying : in a current of warm air.
Wherever results for a particular characteristic are reported, Detection : spray with a mixture of equal volumes of a 2 g/L
the control method must be indicated. solution of 1,3-dihydroxynaphthalene R in ethanol (96 per
The following characteristics may be relevant for titanium cent) R and a 460 g/L solution of sulfuric acid R ; heat at
dioxide used as opacifier in solid oral dosage forms and in 105 °C for 5-10 min.
preparations for cutaneous application. System suitability : the chromatogram obtained with
Particle-size distribution (2.9.31). reference solution (b) shows 3 major spots which are clearly
separated.
Bulk and tapped density (2.9.34).
Results : the principal spot in the chromatogram obtained
with the test solution is similar in position, colour and size
to the principal spot in the chromatogram obtained with
01/2008:0645 reference solution (a).
corrected 6.2
C. Dissolve about 5 mg in 5 mL of water R. Add 5 mL of a 1 g/L
solution of ninhydrin R in ethanol (96 per cent) R and heat
TOBRAMYCIN in a water-bath for 3 min. A violet-blue colour develops.
TESTS
Tobramycinum
pH (2.2.3): 9.0 to 11.0.
Dissolve 1.0 g in 10 mL of carbon dioxide-free water R.
Specific optical rotation (2.2.7) : + 138 to + 148 (anhydrous
substance).
Dissolve 1.00 g in water R and dilute to 25.0 mL with the same
solvent.
Related substances. Liquid chromatography (2.2.29).
Test solution (a). Dissolve 25.0 mg of the substance to be
examined in the mobile phase and dilute to 25.0 mL with the
mobile phase.
C18H37N5O9 Mr 467.5 Test solution (b). Dilute 10.0 mL of test solution (a) to 100.0 mL
[32986-56-4] with the mobile phase.
Reference solution (a). Dissolve 25.0 mg of tobramycin CRS in
DEFINITION the mobile phase and dilute to 100.0 mL with the mobile phase.
4-O-(3-Amino-3-deoxy-α-D-glucopyranosyl)-2-deoxy-6-O-(2,6- Reference solution (b). Dilute 1.0 mL of reference solution (a)
diamino-2,3,6-trideoxy-α-D-ribo-hexopyranosyl)-L-streptamine. to 100.0 mL with the mobile phase.
Substance produced by Streptomyces tenebrarius or obtained Reference solution (c). Dilute 1.0 mL of reference solution (a)
by any other means. to 50.0 mL with the mobile phase.
Content : 97.0 per cent to 102.0 per cent (anhydrous substance). Reference solution (d). Dissolve 10.0 mg of kanamycin B
sulfate CRS in 20.0 mL of the mobile phase. To 1.0 mL of this
PRODUCTION solution, add 2.0 mL of reference solution (a) and dilute to
It is produced by methods of manufacture designed to eliminate 10.0 mL with the mobile phase.
or minimise substances lowering blood pressure. Reference solution (e). Dilute 10.0 mL of reference solution (a)
CHARACTERS to 25.0 mL with the mobile phase.
Appearance : white or almost white powder. Column :
— size : l = 0.25 m, Ø = 4.6 mm ;
Solubility : freely soluble in water, very slightly soluble in
ethanol (96 per cent). — stationary phase : styrene-divinylbenzene copolymer R
(8 μm) with a pore size of 100 nm ;
IDENTIFICATION — temperature : 55 °C.
First identification : A. Mobile phase : mixture prepared with carbon dioxide-free
Second identification : B, C. water R containing 52 g/L of anhydrous sodium
A. Nuclear magnetic resonance spectrometry (2.2.33). sulfate R, 1.5 g/L of sodium octanesulfonate R, 3 mL/L of
tetrahydrofuran R stabilised with butylhydroxytoluene R,
Preparation : 100 g/L solution in deuterium oxide R. and 50 mL/L of 0.2 M potassium dihydrogen phosphate R
Comparison : 100 g/L solution of tobramycin CRS in previously adjusted to pH 3.0 with dilute phosphoric acid R.
deuterium oxide R. Degas.
B. Thin-layer chromatography (2.2.27). Flow rate : 1.0 mL/min.
Test solution. Dissolve 20 mg of the substance to be Post-column solution : carbonate-free sodium hydroxide
examined in water R and dilute to 5 mL with the same solution R diluted 25-fold with carbon dioxide-free water R,
solvent. which is added pulselessly to the column effluent using a
Reference solution (a). Dissolve 20 mg of tobramycin CRS 375 μL polymeric mixing coil.
in water R and dilute to 5 mL with the same solvent. Flow rate : 0.3 mL/min.
Reference solution (b). Dissolve 4 mg of neomycin Detection : pulsed amperometric detector or equivalent with
sulfate CRS and 4 mg of kanamycin monosulfate CRS in a gold working electrode, a silver-silver chloride reference
1 mL of reference solution (a). electrode and a stainless steel auxiliary electrode which is the
Plate : TLC silica gel plate R. cell body, held at respectively + 0.05 V detection, + 0.75 V
oxidation and − 0.15 V reduction potentials, with pulse
Mobile phase : methylene chloride R, concentrated durations according to the instrument used. The temperature
ammonia R, methanol R (17:33:50 V/V/V). of the detector is set at 35 °C.
Application : 5 μL. NOTE : to prevent problems due to salt precipitation, the
Development : over 2/3 of the plate. electrochemical cell can be flushed with water R overnight.

General Notices (1) apply to all monographs and other texts 3103
all-rac-α-Tocopherol EUROPEAN PHARMACOPOEIA 7.0

Injection : 20 μL using a refrigerated injector (4-8 °C) ; inject

test solution (a) and reference solutions (b), (c) and (d).
Run time : 1.5 times the retention time of tobramycin.
Relative retention with reference to tobramycin
(retention time = about 18 min) : impurity C = about 0.35 ;
impurity B = about 0.40, impurity A = about 0.70.
System suitability :
— resolution : minimum 3.0 between the peaks due to B. R = H : 2-deoxy-4-O-(2,6-diamino-2,3,6-trideoxy-α-D-ribo-
impurity A and to tobramycin in the chromatogram hexopyranosyl)-D-streptamine (nebramine),
obtained with reference solution (d) ; if necessary, adjust
the concentration of sodium octanesulfonate in the mobile C. R = OH : 2-deoxy-4-O-(2,6-diamino-2,6-dideoxy-α-D-
phase ; glucopyranosyl)-D-streptamine (neamine).
— signal-to-noise ratio : minimum 10 for the principal peak in
the chromatogram obtained with reference solution (b).
01/2008:0692
Limits : corrected 7.0
— any impurity : not more than twice the area of the principal
peak in the chromatogram obtained with reference all-rac-α-TOCOPHEROL
solution (c) (1.0 per cent) and not more than 1 such peak has
an area greater than the area of the principal peak in the
chromatogram obtained with reference solution (c) (0.5 per int-rac-α-Tocopherolum
cent) ;
— total : not more than 3 times the area of the principal peak
in the chromatogram obtained with reference solution (c)
(1.5 per cent) ;
— disregard limit : the area of the principal peak in the
chromatogram obtained with reference solution (b) (0.25 per
cent). C29H50O2 Mr 430.7
[10191-41-0]
2-Methyl-1-propanol (2.4.24, System B) : maximum 1.0 per
cent m/m. DEFINITION
Water (2.5.12) : maximum 8.0 per cent, determined on 0.30 g. all-rac-2,5,7,8-Tetramethyl-2-(4,8,12-trimethyltridecyl)-3,4-
Sulfated ash (2.4.14) : maximum 0.3 per cent, determined on dihydro-2H-1-benzopyran-6-ol.
1.0 g. Content : 96.0 per cent to 102.0 per cent.
Bacterial endotoxins (2.6.14) : less than 2.0 IU/mg, if intended CHARACTERS
for use in the manufacture of parenteral preparations without
a further appropriate procedure for the removal of bacterial Appearance: clear, colourless or yellowish-brown, viscous, oily
endotoxins. liquid.
Solubility : practically insoluble in water, freely soluble in
acetone, in anhydrous ethanol, in methylene chloride and in
ASSAY
fatty oils.
Liquid chromatography (2.2.29) as described in the test for
related substances with the following modifications. IDENTIFICATION
First identification : A, B.
Injection : test solution (b) and reference solution (e).
Second identification : A, C.
Calculate the percentage content of tobramycin.
A. Optical rotation (2.2.7) : − 0.01° to + 0.01°.
Dissolve 2.50 g in anhydrous ethanol R and dilute to
STORAGE 25.0 mL with the same solvent.
If the substance is sterile, store in a sterile, airtight, tamper-proof B. Infrared absorption spectrophotometry (2.2.24).
container.
Comparison : α-tocopherol CRS.
C. Thin-layer chromatography (2.2.27).
IMPURITIES
Test solution. Dissolve 10 mg of the substance to be
examined in 2 mL of cyclohexane R.
Reference solution. Dissolve 10 mg of α-tocopherol CRS in
2 mL of cyclohexane R.
Plate : TLC silica gel F254 plate R.
Mobile phase : ether R, cyclohexane R (20:80 V/V).
Application : 10 μL.
Development : over 2/3 of the plate.
Drying : in a current of air.
Detection : examine in ultraviolet light at 254 nm.
Results : the principal spot in the chromatogram obtained
A. 4-O-(3-amino-3-deoxy-α-D-glucopyranosyl)-2-deoxy-6-O-(2, with the test solution is similar in position and size to the
6-diamino-2,6-dideoxy-α-D-glucopyranosyl)-L-streptamine principal spot in the chromatogram obtained with the
(kanamycin B), reference solution.

3104 See the information section on general monographs (cover pages)