Teicoplanin

(Ph Eur monograph 2358)

Glycopeptide antibacterial.

Ph Eur

Mixture of glycopeptides produced by certain strains of Actinoplanes teichomyceticus sp.; the 6 principal components of the mixture are teicoplanin A_2-1 to A_2-5 and teicoplanin A_3-1.

Fermentation product.

Minimum 900 IU/mg (anhydrous and sodium chloride-free substance).

Yellowish, amorphous powder.

Freely soluble in water, sparingly soluble in dimethylformamide, practically insoluble in ethanol (96 per cent V/V).

A.  Infrared absorption spectrophotometry (2.2.24).

Comparison  teicoplanin for identification CRS.

B.  Examine the chromatograms obtained in the test for composition and related substances.

Results  The principal peaks (teicoplanins A_3-1, A_2-1, A_2-2, A_2-3, A_2-4 and A_2-5) in the chromatogram obtained with the test solution are similar in retention time and size to the principal peaks in the chromatogram obtained with reference solution (a).

The solution is clear (2.2.1) and not more intensely coloured than reference solution BY₃ or B₄ (2.2.2, Method I).

Dissolve 0.8 g in 10 ml of water R.

6.5 to 7.5.

Dissolve 0.50 g in carbon dioxide-free water R and dilute to 10 ml with the same solvent.

Liquid chromatography (2.2.29): use the normalisation procedure.

Test solution  Dissolve 0.100 g of the substance to be examined in water R and dilute to 50.0 ml with the same solvent.

Reference solution (a)  Dissolve 20 mg of teicoplanin for identification CRS in water R and dilute to 10.0 ml with the same solvent.

Reference solution (b)  Dilute 1.0 ml of reference solution (a) to 10.0 ml with water R. Dilute 1.0 ml of this solution to 20.0 ml with water R.

Reference solution (c)  Dissolve 50.0 mg of mesityl oxide CRS in water R and dilute to 25.0 ml with the same solvent. Dilute 1.0 ml of the solution to 10.0 ml with water R. Dilute 1.0 ml of this solution to 100.0 ml with water R.

• — size: l = 0.25 m, Ø = 4.6 mm;

• — stationary phase: spherical end-capped octadecylsilyl silica gel for chromatography R (5 µm).

• — mobile phase A: mix 900 ml of a 3.0 g/l solution of anhydrous sodium dihydrogen phosphate R, adjusted to pH 6.0 with 1 M sodium hydroxide, and 100 ml of acetonitrile R;

• — mobile phase B: mix 300 ml of a 3.0 g/l solution of anhydrous sodium dihydrogen phosphate R, adjusted to pH 6.0 with 1 M sodium hydroxide, and 700 ml of acetonitrile R;

Flow rate  2.3 ml/min.

Detection  Spectrophotometer at 254 nm.

Injection  20 µl.

Identification  Use the chromatogram supplied with teicoplanin for identification CRS and the chromatogram obtained with reference solution (a) to identify the groups and impurities.

Relative retention of groups and impurities with reference to teicoplanin A_2-2:

• — teicoplanin A₃ group ≤ 0.70;

• — teicoplanin A₂ group > 0.70 and ≤ 1.25 and within this group:

• — teicoplanin A_2-1 group << 1;

• — teicoplanin A_2-2  =  1;

• — teicoplanin A_2-3 group > 1 and < 1.12;

• — teicoplanin A_2-4  =  about 1.12;

• — teicoplanin A_2-5 group > 1.12 and ≤ 1.25;

• — impurities >> 1.25.

Relative retention of principal peaks of the groups with reference to teicoplanin A_2-2 (retention  time  =  about  18  min): teicoplanin  A_3-1  =  about  0.43; teicoplanin  A_2-1  =  about  0.93; teicoplanin  A_2-3  =  about  1.04; teicoplanin  A_2-4  =  about  1.12; teicoplanin  A_2-5  =  about  1.14.

System suitability  Reference solution (a):

• — the chromatogram obtained is similar to the chromatogram supplied with teicoplanin for identification CRS;

• — resolution: minimum 1.0 between the peaks due to teicoplanin A_2-4 and teicoplanin A_2-5.

Calculate the percentage content of the different components using the following equations:

• — teicoplanin A₂ group: minimum 80.0 per cent;

• — teicoplanin A_2-1 group: maximum 20.0 per cent;

• — teicoplanin A_2-2: 35.0 to 55.0 per cent;

• — teicoplanin A_2-3 group: maximum 20.0 per cent;

• — teicoplanin A_2-4: maximum 20.0 per cent;

• — teicoplanin A_2-5 group: maximum 20.0 per cent;

• — teicoplanin A₃ group: maximum 15.0 per cent;

• — total of impurities other than mesityl oxide with a retention time more than 1.25: maximum 5.0 per cent;

• — disregard limit: the area of the peak due to teicoplanin A_2-2 in the chromatogram obtained with reference solution (b) (0.25 per cent).

Maximum 5.0 per cent, expressed as sodium chloride (anhydrous substance).

Dissolve 1.000 g in 300 ml of water R, stir and acidify with 2 ml of nitric acid R. Titrate with 0.1 M silver nitrate, determining the end-point potentiometrically (2.2.20).

1 ml of 0.1 M silver nitrate is equivalent to 5.844 mg of NaCl.

Maximum 20 ppm.

0.50 g complies with test G. Prepare the reference solution using 100 µl of lead standard solution (100 ppm Pb) R. Filter the solutions through a membrane filter (nominal pore size 0.45 µm).

Liquid chromatography (2.2.29) as described under the test for composition and related substances with the following modifications.

Injection  20 µl of the test solution and reference solution (c).

Relative retention  With reference to teicoplanin A_2-2 (retention  time  =  about  18  min): impurity  A  =  about  0.6.

• — impurity A: maximum twice the area of the principal peak in the chromatogram obtained with reference solution (c) (0.2 per cent).

Maximum 15.0 per cent, determined on 0.300 g.

Less than 0.31 IU/mg.

Carry out the microbiological assay of antibiotics (2.7.2), using the diffusion method. Use teicoplanin CRS as the reference substance.

Protected from light, at a temperature of 2  °C to 8  °C.

Specified impurities:  A.

A.  4-methylpent-3-en-2-one (mesityl oxide).

Ph Eur