ANTIBIOTIC RESIDUE ANALYSIS IN EDIBLE MEAT USING HIGH PERFORMANCE LIQUID CHROMATOGRAPHY By Betty Kerubo I56/76366/2014 Supervisors: Dr. Gabriel Aboge Dr. George Obiero

Introduction Administration of antibiotics has facilitated successful management of bacterial infections. Today with the advancement in technology their use has been vast in growth promotion. Antibiotic efficacy is dependent on judicious and appropriate administration otherwise residual in food producing animals. There is evidence of the existence of residual antibiotics in food producing animals.

Introduction… Low antibiotic residue levels in high income regions such as Europe recording ˂ 1% of reported cases (Food & Authority, 2013). The food safety inspection service monitoring plan 2013 reported about 0.13% residues in meat of healthy animals (Are et al., 2013). Samples analyzed for antibiotic residues in meat in Nairobi slaughter houses in Kenya reported 20% tetracycline (Muriuki et al., 2001). Despite this trace antibiotic levels recorded by regulatory agencies, the rate of consumption will lead to accumulation over time and adverse effects (Boeckel et al., 2014).

Introduction… Allergic reactions in sensitive individuals are few consequences of residual antibiotics. This problem is recurrent in countries with limited regulation agencies and lack of awareness by regulatory bodies (Bedada et al., 2012). Resistance arising from residual antibiotics will burden the human population to discard existent antibiotics for newer ones which tend to be expensive (Boeckel et al., 2014). Regulatory agencies such as WHO and FDA have made efforts to manage these residues setting MRLs for various antibiotics (Nisha, 2008).

Problem statement Antibiotic residues are as a result of inappropriate antibiotic use and lack of awareness by regulatory bodies. Research has been done in Kenya to analyse the levels of antibiotic residues but still this data is inadequate. Trends in antibiotic use have seen increments 36% between the years 2000 to 2010(Fao, Workshop, & Drugs, n.d.). Low and middle income countries account for about 76% of this, with last resort antibiotic accounting for 58% Research to establish the quantities of residual antibiotics needs to be addressed to provide supplement the already existing data.

Hypothesis There are higher antibiotic residue levels in edible meat from Kenyan slaughter houses compared to the maximum residue limits (MRLs) set by WHO/FDA.

Objectives General objective To determine the presence of oxytetracycline streptomycin and diminazene in kidney, liver and other edible meat. Specific objectives To monitor levels of streptomycin, oxytetracycline and diminazene in kidney, liver and other edible meat; To quantify the amount of streptomycin, diminazene and oxytetracycline; To analyze contraindications associated with residual antibiotics to enhance prospect development of consensus and judicious antibiotic residue tolerant levels

Justification of the study Findings from this study will Provide the current quantities of residual antibiotics in edible meat; Assess the extent of hazard presented to the consumers of food producing animals.

Expected outcomes Finding from this study will Help to possibly to suggest and recommend new MRLs to regulatory agencies; Help to prioritize programs that will ensure judicious use of antimicrobials through concerted efforts by the international community.

Materials and Methods Sample collection Meat extraction (Resources & State, 2010) Meat juice HPLC clean up Oxytetracycline (Mahmoudi et al., 2014). Streptomycin (Zeina, Pamela, & Fawwak, 2013) Diminazene (An, 2013) HPLC data analysis

Statistical analysis For H 0 : ρ = Ɵ VS H 1 : ρ Ɵ z c = Ƶ - µ Ƶ δ Ƶ Where Ƶ = ½ ln (1+r) (1- r) µ Ƶ = log (1+ ρ 0 ) (1 –ρ 0 ) δ Ƶ = 1 1 √(n-3) if z c ˂ Ƶ α accept H 0 at 0.05% L.O.S

Timeline Activity September 2015 October 2015 November 2015 December 2015 January 2016 February 2016 March 2016 April 2016 Proposal development Sample collection and Meat extraction Meat HPLC clean up Data analysis Thesis write up and submission

Budget ItemQuantity Cost Acetonitrile hplc grade /= 32500/= Methanol hplc grade /= 15000/= Nylon membrane filters0.45mm by 47mm 10000/= Nylon syringe filters acrodiscs /= 5ml plastic pipette (disposable)400 30/= 12000/= 1.5 mls autosampler 1000/= 30000/= Hplc grade water6500/= Whatman filter papers (no. 1) /= 2 mls Syringes hypodermic500 5/= 2500/= 10 mls Syringes500 10/= 5000/= Nitrogen gas120M3 7500/= Blue micropipete tips2 1000/= 2000/= yellow micropipete tips2 1000/= 2000/= White /clear micropipete tips2 1500/= 3000/= 15 mls Centrifuge tubes PPE300 25/= 7500/= Paper towels8 300/= 2400/= Sub total /= Contigency (10% of sub total) 17140/= Grand total (contigency +sub total) /=

Acknowledgement Dr. Gabriel Aboge Dr. George Obiero University of Nairobi ILRI