Penicillin G (PG) is widely used for therapeutic or prophylactic purposes to treat cattle diseases. PG might be applied in the form of procaine salt to achieve longer lasting activity. PG procaine (PGp) is a combination of PG with the local anaesthetic agent, procaine, and is slowly absorbed into the circulation and hydrolysed to PG. It is used where prolonged low concentrations of PG are required.

The Codex (Codex Alimentarium Commission) has set a maximum residue limit (MRL) for PG (including PGp) in beef (cattle muscle) at 0.05 μg/g to ensure that beef products for human consumption are virtually residue-free of these substances.¹ The acceptable (or ideal) method for the routine monitoring of veterinary drugs in foods of animal origin must be simple, quick, economical in terms of time and cost, and cause negligible harm to the environment and analyst.

Eliminating the use of toxic organic solvents and reagents is an important goal in terms of environmental conservation, human health and the economy. Although high performance liquid chromatography (HPLC) techniques with ultraviolet (UV) detection to simultaneously determine PG and PGp in plasma² or serum³ have been reported, there is no method that does not use toxic solvents/reagents. This article describes a rapid and inexpensive technique that does not use hazardous chemicals to monitor PG and PGp residues in beef.

Reagents and apparatus: Penicillin G potassium (PG) and penicillin G procaine (PGp) standards were purchased from Wako Pure Chem. (Osaka, Japan) and Nippon Zenyaku Kyogyo (Koriyama, Japan), respectively. Ethanol (purity 99.5%) and distilled water were of HPLC grade (Wako). Stock standard solutions of PG and PGp were prepared by dissolving each of the compounds in water to a concentration of 100 μg/mL. Working mixed standard solutions of these compounds were prepared by diluting the stock solutions with water. A handheld ultrasonic-homogenizer (model HOM-100, 2 mm i.d. probe, Iwaki Glass, Funabashi, Japan), a micro-centrifuge (Biofuge fresco, Kendo Lab. Products, Hanau, Germany), and an Ultrafree-MC/PL (Millipore, Bedford, Massachusetts, USA) as a centrifugal ultra-filtration unit were used in the sample preparation.

HPLC: The HPLC system included a model PU-980 pump and DG-980-50 degasser (both from Jasco Corp., Tokyo, Japan), as well as a model SPD-M10A VP photo-diode array (PDA) detector (Shimadzu Scientific Instruments, Kyoto, Japan). The analytical column was an Inertsil WC300C4 (100 × 4.6 mm, 5 μm) column (GL Science, Tokyo, Japan). The isocratic mobile phase was 0.04 mol/L phosphoric acid (pH 7.0)–ethanol (8:2, v/v) and the flow-rate was 1.0 mL/min. PDA detector was scanned from 190–350 nm: detecting at 205 nm for PG and 290 nm for PGp, respectively (a maximum UV absorption for each compound). The column temperature was operated at 40 °C The injection volume was 20 μL.

Sample preparation: An accurately weighed 0.1 g homogenized beef sample was placed in a micro-centrifuge tube and homogenized with the ultrasonic homogenizer for 30 s with 0.6 mL of ethanol. After being homogenized, the capped tube was centrifuged at 12000 g for 5 min. A 50 μL portion of supernatant liquid was placed into an Ultrafree-MC/PL and centrifuged at 5000 g for 5 min. The ultra-filtrate was injected into an HPLC system.

Recovery test: The recoveries of PG and PGp from blank beef samples spiked at 0.05, 0.1 and 0.2 μg/g, respectively, were determined. These fortification levels were prepared by adding 10 μL of three working mixed standard solutions, 10, 20 and 40 μg/mL, respectively, to separate 2 g portions of the samples. Fortified samples were allowed to stand at 4 °C for 24 h after the mixed standard addition and then mixed prior to the test.