J. vet. Pharmacol. Therap. 23, 397–399, 2000. SHORT COMMUNICATION Determination of thiamphenicol residues in albumin and yolk of hen eggs M. GIORGI* M. ROMANI* M. BAGLIACCA † & G. MENGOZZI* *Department of Veterinary Clinics, Pharmacology and Toxicology Section, † Department of Animal Production, Faculty of Veterinary Medicine, University of Pisa, V.le delle Piagge 2, 56124 Pisa, Italy (Paper received 3 September 1999; accepted for publication 21 April 2000) Thiamphenicol is a structural analogue of chloramphenicol with a broad spectrum and similar mechanism of action, which has been shown to be valuable for the treatment of bacterial infections in both animals and humans (Laplassotte and Brunaud, 1961; Yunis et al., 1973; Abdennebi, 1991). Thi- amphenicol has a greater in vivo activity against pathogenic bacteria than other structural analogues and it is also active against some bacteria that are resistant to chloramphenicol. However, in spite of their chemical similarity, the toxicity of thiamphenicol is lower (reversible dose-related bone marrow suppression) (Yunis et al., 1973; Rankin, 1975). The presence of thiamphenicol residues in food-producing animals is undesirable from the standpoint of human safety, therefore it was important to develop sensitive methods for its determination. Various methods have been described in the literature for thiamphenicol residue evaluation in several ani- mal species: chickens (Nagata and Saeki, 1991), beef and dairy cattle (Abdennebi et al., 1994), laying hens (Romani et al. 1999) and fish (Nagata and Saeki, 1992), but for the determi- nation of thiamphenicol residues in eggs, a sensitive and specific analytical method is required which will separate it from other egg components. The aim of the present study was to develop a rapid, sensitive and specific high performance liquid chromato- graphic method (HPLC) and to investigate thiamphenicol residues in egg albumin and yolk after administering single and multiple oral doses to laying hens. Twenty 6-month-old Isabrown laying hens, weighing 1.6 – 2.00 kg, were individually weighed, randomly housed in seven numbered cages (with water and food available ad libitum) such that there were six cages of three animals and one of two animals. Animal care and handling were performed according to the provisions of the EC Council Directive 86/609 EEC, recognized and adopted by the Italian Government (DL 27/1/ 1992, no. 116). On the day of the treatment, the birds were allocated into two groups of twelve and eight hens, respectively. Capsules containing a dose of 40 mg/kg of thiamphenicol base (as the glycinate form, Glitisol ® , Zambon, Milan, Italy) were prepared for each hen and administered p.o. to the first group (cages 1–4), while the second group (cages 5–7) re- ceived the same oral dose on each of 5 successive days. Eggs (shell colored) were collected daily for 15 days (total number of eggs 294); yolk and albumin were separated, weighed and stored at -20 °C. The shells were weighed after washing and 24 h drying at room temperature (20 °C, 60% relative humidity). Shell thick- ness (with shell membranes) was measured by micrometer (precision 0.01 mm) on acute, obtuse and equatorial regions. Eggs were collected from five untreated laying hens for blank controls and for preparation of spiked samples. Sample preparation and drug extraction were carried out according to a method developed previously and partially modified (Romani et al., 1999). Thiamphenicol standards were prepared from pooled blank yolk and albumin by adding known amounts of thiamphenicol to achieve concentrations ranging from 0 to 5 g/g. Two grammes of albumin or yolk samples were extracted with 6 mL of a phosphate buffer (monobasic potassium phosphate solution 67 mM and anhydrous di-sodium hydrogen orthophosphate solution 67 mM, mixed in a ratio of 4:6 v/v immediately before use) and 20 mL of ethyl acetate. Samples were shaken in a horizontal mixer for 30 min and centrifuged at 5000 ×g for 15 min. The organic phase was collected and the hydrophilic phase extracted again with 10 mL of ethyl acetate. The total organic phase was evaporated to dryness in a 40°C rotavapor and the dry residue was dissolved in 1 mL of HPLC mobile phase, which was a mixture of acetate buffer (acetic acid 0.01 M and sodium acetate 0.01 M in double-distilled water) and acetonitrile (8:2 v/v) and 20 L of this sample were injected into a Jasco HPLC system using a Spherisorb C18 ODS2 column for separation. Ultraviolet ab- sorbance ( =230 nm) was used for drug detection. The reten- tion time for thiamphenicol was 6.30 min and the quantification limit of the method was 10 g/kg. Extraction recoveries of thiamphenicol from yolk and albu- min were 75.2 2.0 and 80.4 1.1%, respectively. The preci- sion of the method was expressed as the coefficient of variation © 2000 Blackwell Science Ltd 397