(Tiamulin for Veterinary Use, Ph. Eur. monograph 1660)
C28H47NO4S 493.8 55297-95-5
Action and use
Antibacterial.
DEFINITION
(3aS,4R,5S,6S,8R,9R,9aR,10R)-6-Ethenyl-5-hydroxy-4,6,9,10-tetramethyl-1-oxodecahydro-3a,9-propanocyclopenta[8]annulen-8-yl [[2-(diethylamino)ethyl]sulfanyl]acetate.
Semi-synthetic product derived from a fermentation product.
Content
96.5 per cent to 102.0 per cent (dried substance).
CHARACTERS
Appearance
Sticky, translucent yellowish mass, slightly hygroscopic.
Solubility
Practically insoluble in water, very soluble in methylene chloride, freely soluble in anhydrous ethanol.
IDENTIFICATION
Infrared absorption spectrophotometry (2.2.24).
Comparison: Ph. Eur. reference spectrum of tiamulin.
TESTS
Appearance of solution
The solution is clear (2.2.1) and its absorbance (2.2.25) at 420 nm is not greater than 0.050.
Dissolve 2.5 g in 50 mL of methanol R.
Related substances
Liquid chromatography (2.2.29).
Ammonium carbonate buffer solution pH 10.0 Dissolve 10.0 g of ammonium carbonate R in water for chromatography R, add 22 mL of perchloric acid solution R and dilute to 1000.0 mL with water for chromatography R. Adjust to pH 10.0 with concentrated ammonia R1.
Solvent mixture acetonitrile R1, ammonium carbonate buffer solution pH 10.0 (50:50 V/V).
Test solution: Dissolve 0.200 g of the substance to be examined in the solvent mixture and dilute to 50.0 mL with the solvent mixture.
Reference solution (a): Dissolve 0.250 g of tiamulin hydrogen fumarate CRS in the solvent mixture and dilute to 50.0 mL with the solvent mixture.
Reference solution (b): Dilute 1.0 mL of the test solution to 100.0 mL with the solvent mixture.
Reference solution (c): Dilute 0.1 mL of toluene R to 100 mL with acetonitrile R. Dilute 0.1 mL of this solution to 100.0 mL with the solvent mixture.
Column:
— size: l = 0.15 m, Ø = 4.6 mm;
— stationary phase: end-capped octadecylsilyl silica gel for chromatography R (5 μm);
— temperature: 30 °C.
Mobile phase: acetonitrile R1, ammonium carbonate buffer solution pH 10.0, methanol R2 (21:30:49 V/V/V).
Flow rate: 1.0 mL/min.
Detection: Spectrophotometer at 212 nm.
Injection: 20 μL.
Run time: 3 times the retention time of tiamulin.
Relative retention: With reference to tiamulin (retention time = about 18 min): impurity A = about 0.22; impurity B = about 0.5; impurity C = about 0.66; impurity D = about 1.1; impurity F = about 1.6; impurity E = about 2.4.
System suitability: Reference solution (a):
— baseline separation between the peaks due to tiamulin and impurity D.
Limits:
— impurities A, B, C, D, E, F: for each impurity, not more than the area of the principal peak in the chromatogram obtained with reference solution (b) (1.0 per cent);
— any other impurity: for each impurity, not more than 0.2 times the area of the principal peak in the chromatogram obtained with reference solution (b) (0.2 per cent);
— total: not more than 3 times the area of the principal peak in the chromatogram obtained with reference solution (b) (3.0 per cent);
— disregard limit: 0.1 times the area of the principal peak in the chromatogram obtained with reference solution (b) (0.1 per cent); disregard any peak present in the chromatogram obtained with reference solution (c).
Loss on drying (2.2.32)
Maximum 1.0 per cent, determined on 1.000 g by drying in an oven at 80 °C.
Bacterial endotoxins (2.6.14, Method D)
Less than 0.4 IU/mg, determined in a 1 mg/mL solution in anhydrous ethanol R (endotoxin free) diluted 1:40 with water for bacterial endotoxins test.
ASSAY
Liquid chromatography (2.2.29) as described in the test for related substances with the following modification.
Injection: Test solution and reference solution (a).
Calculate the percentage content of C28H47NO4S, from the declared content of tiamulin hydrogen fumarate CRS.
STORAGE
Protected from light.
IMPURITIES
Specified impurities A, B, C, D, E, F.
Other detectable impurities (the following substances would, if present at a sufficient level, be detected by one or other of the tests in the monograph. They are limited by the general acceptance criterion for other/unspecified impurities and/or by the general monograph Substances for pharmaceutical use (2034). It is therefore not necessary to identify these impurities for demonstration of compliance. See also 5.10. Control of impurities in substances for pharmaceutical use) G, H, I, J, K, L, M, N, O, P, Q, R.
A. (3aS,4R,5S,6S,8R,9R,9aR,10R)-6-ethenyl-5,8-dihydroxy-4,6,9,10-tetramethyloctahydro-3a,9-propanocyclopenta[8]annulen-1(4H)-one (mutilin),
B. 2-(benzylsulfanyl)-N,N-diethylethan-1-amine,
C. 2,2′-disulfanediylbis(N,N-diethylethan-1-amine),
D. (1aS,1bR,2R,3R,5S,6S,7R,7aS,8aR,11R)-5-ethenyl-6-hydroxy-2,5,7,11-tetramethyldecahydro-2,7a-propanocycloocta[3,4]cyclopenta[1,2-b]oxiren-3-yl [[2-(diethylamino)ethyl]sulfanyl]acetate,
E. (3aS,4R,6S,8R,9R,9aR,10R)-6-ethenyl-4,6,9,10-tetramethyl-1,5-dioxodecahydro-3a,9-propanocyclopenta[8]annulen-8-yl [[2-(diethylamino)ethyl]sulfanyl]acetate (11-oxotiamulin),
F. (1Ξ,3aR,4R,6S,8R,9R,9aR,10R)-6-ethenyl-1-hydroxy-4,6,9,10-tetramethyl-5-oxodecahydro-3a,9-propanocyclopenta[8]annulen-8-yl [[2-(diethylamino)ethyl]sulfanyl]acetate (1-hydroxy-11-oxotiamulin),
G. (3aS,4R,5S,6S,8R,9R,9aR,10R)-6-ethenyl-5-hydroxy-4,6,9,10-tetramethyl-1-oxodecahydro-3a,9-propanocyclopenta[8]annulen-8-yl hydroxyacetate (pleuromutilin, pleuromulin),
H. (2E)-4-[(2Ξ)-2-[(3aS,4R,5S,6R,8R,9R,9aR,10R)-8-[[[[2-(diethylamino)ethyl]sulfanyl]acetyl]oxy]-5-hydroxy-4,6,9,10-tetramethyl-1-oxodecahydro-3a,9-propanocyclopenta[8]annulen-6-yl]-2-hydroxyethoxy]-4-oxobut-2-enoic acid (19,20-dihydroxytiamulin 20-fumarate),
I. (2E)-4-[[(1Ξ,2Ξ,3aS,4R,5S,6S,8R,9R,9aR,10R)-8-[[[[2-(diethylamino)ethyl]sulfanyl]acetyl]oxy]-6-ethenyl-1,5-dihydroxy-4,6,9,10-tetramethyldecahydro-3a,9-propanocyclopenta[8]annulen-2-yl]oxy]-4-oxobut-2-enoic acid (2,3-dihydroxytiamulin 2-fumarate),
J. (3aS,4R,5S,6S,8R,9R,9aR,10R)-6-ethenyl-5-hydroxy-4,6,9,10-tetramethyl-1-oxodecahydro-3a,9-propanocyclopenta[8]annulen-8-yl acetate (mutilin 14-acetate),
K. (3aS,4R,5S,6S,8R,9R,9aR,10R)-6-ethenyl-8-hydroxy-4,6,9,10-tetramethyl-1-oxodecahydro-3a,9-propanocyclopenta[8]annulen-5-yl acetate (mutilin 11-acetate),
L. (3aS,4R,5S,6S,8R,9R,9aR,10R)-6-ethenyl-5-hydroxy-4,6,9,10-tetramethyl-1-oxodecahydro-3a,9-propanocyclopenta[8]annulen-8-yl [(4-methylbenzene-1-sulfonyl)oxy]acetate (pleuromutilin 22-tosylate),
M. (3aS,4R,5S,6S,8R,9R,9aR,10R)-6-ethenyl-4,6,9,10-tetramethyl-1-oxodecahydro-3a,9-propanocyclopenta[8]annulen-5,8-diyl diacetate (mutilin 11,14-diacetate),
N. (2E)-4-[2-[[(3aS,4R,5S,6S,8R,9R,9aR,10R)-6-ethenyl-5-hydroxy-4,6,9,10-tetramethyl-1-oxodecahydro-3a,9-propanocyclopenta[8]annulen-8-yl]oxy]-2-oxoethoxy]-4-oxobut-2-enoic acid (pleuromutilin 22-fumarate),
O. 2-(diethylamino)ethane-1-thiol,
P. (3aS,4R,5S,6S,8R,9R,9aR,10R)-6-ethenyl-5-hydroxy-4,6,9,10-tetramethyl-1-oxodecahydro-3a,9-propanocyclopenta[8]annulen-8-yl [(benzenesulfonyl)oxy]acetate,
Q. (2Ξ,3aS,4R,5S,6S,8R,9R,10R)-6-ethenyl-2,5-dihydroxy-4,6,9,10-tetramethyl-2,3,4,5,6,7,8,9-octahydro-3a,9-propanocyclopenta[8]annulen-8-yl [[2-(diethylamino)ethyl]sulfanyl]acetate (3,4-didehydro-2-hydroxytiamulin),
R. N-benzyl-N,N-dibutylbutan-1-aminium.
