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affect the natural soil community and also disturb soil function. In addition, it was
found that wastewater and run-off from agricultural land are mainly responsible for
the contamination of aquatic systems (Alder, 2001).
For this purpose, it was necessary to establish a simple and reliable method for
extraction, separation and determination of veterinary pharmaceuticals in soil. The
drugs investigated included three sulfonamides (sulfaguanidine, sulfadiazine and
sulfamethazine), sulfonamide synergist (trimethoprim), tetracycline (oxytetracycline),
fluoroquinolone (norfloxacine) and b-lactam (penicillinG/procaine). In this contribution,
we worked out the procedure using ultrasound extraction (USE) of investigated
compounds from soil, and their subsequent thin-layer chromatographic (TLC)
determination.
MATERIALS AND METHODS
Materials
All used chromatographic solvents were p.a. grade (Kemika, Zagreb, Croatia). In
this work, chromatographic plates HPTLC CN F254 10 x 20 cm, (Merck, Darmstadt,
Germany) were used. Soil was collected at Medvednica, a hill near Zagreb. At
least 10 years before collecting, the soil had not been treated with any antibiotics.
Its composition was: sand 61.45%, silt 20.75%, clay 11.80% and organic matter
6.44%.
Standards
Trimethoprim (TMP), oxytetracycline (OTC), norfloxacine (NOR), sulfaguanidine
(SGUA), sulfamethazine (SMETH), sulfadiazine (SDIAZ) and penicillinG/procaine
(PGP) (VETERINA Animal Health Ltd., Kalinovica, Croatia) were min. 99 % pure.
Stock solution of pharmaceutical mixture was prepared by dissolving accurate
quantities of the powdered standards in methanol. Mass concentration of each
pharmaceutical in the mixture was: 5 mg/L for NOR, 100 mg/L for SMETH, SDIAZ,
TMP, OTC and PGP, and 200 mg/L for SGUA. Calibration standards were made by
serial dilution of a stock standard solution with methanol in the working range from
0.15 to 1.5 mg/L for NOR, from 10 to 60 mg/L for OTC, TMP, SDAIZ, SMETH, and
PGP, and from 30 to 110 mg/L for SGUA.
Soil Spiking and Extraction Procedure
Ten millilitres of standard solution was added to 100 g of dried and sieved soil. An
additional 100 mL of methanol was added to cover the soil particles; the slurry
was mixed for 1 h and then left at room temperature for 24 hours to allow solvent
evaporation.
Accurately weighted spiked soil (10 g) was added to 20 mL of methanol and
sonicated for 15 min in an ultrasonic bath (frequency 25–40 Hz, UZ-20R, Iskra, Kranj,
Slovenia). The extracts were filtered through Whatman No.40 filter paper. The filtrate
was evaporated on a rotary vacuum evaporator (R-114/A, Büchi, Switzerland) at 40°
to dryness. The residue was dissolved in 1 mL of methanol and this solution was
directly spotted on a TLC plate.
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