In agreement with EU and Polish legislation, both production process of medicated feedstuffs as well as its usage has to be strictly monitored and controlled. The main aims of the process control are determination of active ingredient concentration, homogeneity of the product and accordance with GMP. Main group of actives consist of antimicrobials such as tetracyclines, tylosin, amoxycyline, tiamulin and sulphonamides.

Tiamulin has higher activity than natural pleuromutilin first extracted in 1951 from Pleurotus mutilus (Fr.) Sacc. and Pleurotus Passeckerianus Pil. It is active against Mycoplasma spp., gram positive bacteria such as streptococci and staphylococci and obligate anaerobes. It is mainly used in treatment of swine (dysentery caused by Brachyspira hyodeysentreiae, bacterial pneumonia caused by Pasteurella multicida) and poultry.

Tiamulin is a semi-synthetic member of pleuromutilin class of antibiotics. Core of the molecule constitute of three cyclic hydrocarbons with 8 asymmetric carbons. Tiamulin base is lipophilic but in premixes it is mainly used as hydrogen fumarate salt freely soluble in water.

Chemical structure of tiamulin, lack of fluorescence,  low UV activity and complexed matrix causes problems in quantitative analysis. Published methods for determination of tiamulin in various materials use thin layer or liquid chromatography as well as gas chromatography, well established is also microbiological assay. The aim of this study was to optimize extraction procedure and HPLC determination method of tiamulin hydrogen fumarate in medicated feedstuffs and premixes available on polish market.

Solid-liquid water extraction of premix samples was used in the procedure. Single or duplicate extraction and different sample size (0.25 – 2.5g) were tested giving recovery over 90%. It was decided to use duplicate extraction of 0.25g sample for further procedures.

1% sodium carbonate solution followed by organic solvent (hexane : ethyl acetate)  extraction was employed to free tiamulin from feed matrix. Next step of the procedure was to bring a portion of extract to dryness and dissolve residue in 0.1% tartaric acid solution. Small sample size of 2.0g were tested giving CV% below 6% between 3 series of experiments.

All extracts were analysed on Agilent 1200 series system equipped with Gemini NX, 4.6x150mm column and UV-Vis detection at 208nm. Methanol : acetonitrile : ammonium carbonate (50:25:25) mixture at flow rate of 1.0mL/min was used as mobile phase. Linearity, specificity, precision and recovery level were tested results indicating applicability of the optimized method for tiamulin determination in premixes and medicated feedstuffs.

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