Test formulations
Injectors containing 600 mg cloxacillin benzathine (formulated as Orbenin Extra Dry Cow, Pfizer Animal Health, Sandwich, UK) were used. Orbenin Extra Dry Cow is approved for intramammary administration to cows at drying off.
Isolated perfused bovine udder
As described in detail by Kietzmann et al. [8], medium sized udders with a symmetric shape and a teat length of 6-8 cm from healthy German Black Pied cows that were lactating prior to slaughter were used. Each cow's udder was examined prior to slaughter by palpation of the quarters and inspection of the milk to check for the absence of clinical mastitis. Within 10 - 15 min post-slaughter, blood clots in the vessels of the glands were cleared using 1 L heparinised Tyrode solution per udder half. Thereafter, the udders were transported for 20 min in a plastic tub to the laboratory. Within a few minutes after arrival at the laboratory, the udder was fixed in a "natural" position to a metal frame using the proximally inserting skin and suspensory ligament. Within a few minutes, the large arteries of the udder were supplied with the perfusion fluid delivered via silicone tubes. The larger veins were also cannulated to allow sampling and removal of the perfusate, whereas the smaller veins were closed using artery forceps. Six front and rear quarters from six udder udders were used.
Each udder half was perfused with 90 - 120 mL/min of Tyrode solution (136.8 mmol/L NaCl, 2.7 mmol/L KCl, 1.8 mmol/L CaCl2 × 2H2O, 1.05 mmol/L MgCl2 × 6H2O, 0.416 mmol/L NaH2PO4 × 2H2O, 11.9 mmol/L NaHCO3 and 5.5 mmol/L D(+)-glucose × 1H2O; 39°C) gassed with carbogen (95% O2, 5% CO2) using a peristaltic pump. The mammary glands were milked over about 5 minutes during a 30 min equilibration phase. The viability of the perfused udders was controlled using biochemical parameters such as lactate dehydrogenase(LDH)-activity, glucose consumption and lactate production in the perfusate [4, 5, 8]. The perfusate flux was about 90-120 mL/minute per udder half.
Study design
After reaching a physiological like status during the equilibration period, one intramammary syringe of Orbenin Extra Dry Cow each was administered via the teat canal and massaged into the glandular cistern of one front and one rear quarter of the same side of the udder. After 6 h, four gland tissue samples were taken from the central region of the treated quarters (front quarter: 4, 8, 12, 16 cm distance to the teat, rear quarter: 5, 10, 15, 20 cm distance to the teat). Perfusate was sampled before and hourly after treatment for six h. Perfusate and tissue samples were stored frozen at -20°C.
Analysis of cloxacillin concentrations
Concentrations of cloxacillin in glandular tissue and in perfusate samples were analysed using a high performance liquid chromatography (HPLC) method.
Tissue extraction. Samples of glandular tissue (1 g) were spiked with 10 mL of an internal standard stock solution [1.0 g/l oxacillin (Sigma, Deisenhofen, Germany) for cloxacillin quantification and filled up to a total volume of 4 mL with Soerensen phosphate buffer [33 mmol/l KH2PO4/Na2HPO4 (Merck, Darmstadt, Germany) at pH 6.8 for cloxacillin extraction. Soerensen phosphate buffer was also used in all further steps of extraction. The tissue was homogenized at 20 000 rpm at 4°C for 90 s (Ultra Turrax®, Janke and Kunkel, Staufen, Germany). After centrifugation at 23000 g at 4°C for 5 min, a supernatant fluid free of visible particles was obtained. For the following fluid-fluid extraction and detection of oxacillin by high pressure liquid chromatography (HPLC), a method of Schadewinkel-Scherkl [13] was modified. 1.5 mL of the prepared samples was added with 3 mL dichloromethane (Merck, Darmstadt, Germany) and immediately after acidifying with 100 μl 0.5 mol/l sulphuric acid was moderately shaken for 20 s. This was followed by centrifugation at 1500 g at 4°C for 10 min. Then 2 mL of the lower organic phase was intensively mixed with 1 mL phosphate buffer for 20 s and centrifuged again and the supernatant was separated for analysis.
HPLC analysis. Sub-samples of 100 μl were injected by an autosampler (Model 508, Beckman, Fullerton, CA, USA) and chromatographed at 40°C on a HPLC column (LiChroCART® 250-4 mm, LiChrospher® 100 RP-18e (5 μm), Merck, Darmstadt, Germany) combined with a guard column (LiChroCART® 4-4 mm, LiChrospher® 100 RP-18e (5 μm), Merck, Darmstadt, Germany). The eluent for cloxacillin analysis [phosphate buffer (33 mmol/l KH2PO4/Na2HPO4, pH 2.6)/acetonitrile (Roth, Karlsruhe, Germany) 66 : 34] was pumped at a flow rate of 1.5 mL/min (Model 126, Beckman, Fullerton, CA, USA). Cloxacillin was detected using ultraviolet light (Model 166, Beckman, Fullerton, CA, USA) at a wavelength of 210 nm. The areas under the curves were directly integrated (Software 32 Karat 5.0, Beckman, Fullerton, CA, USA).
Analysis validation. For the calculation of experimental sample concentrations, untreated udder matrix was spiked with the analyte [5 μg/g tissue]. Calibration curves were in the range of 0.5 to 5 and 5 to 400 μg cloxacillin/g tissue. The spiked samples were extracted and analysed under the same conditions as the experimental samples to create regression lines. The recovery was 91.4 ± 12.4% for cloxacillin. The limit of quantification was 0.56 μg/g. Intraday and interday precision were below 20%.