Analytical grade copper sulphate, citric acid, disodium hydrogen phosphate, potassium-dihydrogen phosphate were purchased from Merck (Germany). Analytical standard of ampicillin, dialysis tubing cellulose membrane was procured from Sigma Aldrich Co, USA.

Copper sulphate solution: Copper sulphate pentahydrate of 3.93 g was dissolved in 1 litre water.

Buffer solution pH 5.2: 0.1 M citric acid solution of 464 ml was mixed with 0.2 M disodium hydrogen phosphate solution of 536 ml. Adjust the pH, if necessary, to 5.2±0.05 with the citric acid or disodium hydrogen phosphate solution. To 15 ml of the copper sulphate solution add the buffer at pH 5.2 to a volume of 1 litre. (1 ml of this solution contains 15 µg of copper).

The study was conducted on clinically presented horses of age group 8-12 years of age with 385-480 kg body weights. Animals were monitored during the collection of blood samples. The protocol of the study was approved by the Committee for the Purpose of Control and Supervision of Experiments on Animals (Order No. F.No.25/4/2019-CPCSEA, dt: 20/03/2019).

Blood samples were collected into heparinised vials from jugular veins. Plasma was separated by centrifugation at 3000 rpm for 15 minutes and stored at -20°C till analysis further.

In vitro binding of ampicillin to plasma proteins was determined by employing the equilibrium dialysis technique (Gupta et al., 2006; Singh et al., 2019). The dialyzing bags (4 Å pore size), 10 cm long were washed in running tap water and soaked overnight in phosphate buffer. Ampicillin concentrations (3.125, 6.25, 12.5, 25, 50, 100 µg/ml) were prepared in pooled plasma separated from blood taken from apparently healthy horses. Each dialyzing bag was knotted at one end before filling 5ml of plasma containing known amount of drug and the other end was then securely tied. Each bag was immersed in separate tubes containing 5 ml of phosphate buffer (0.2 M; pH 7.4; disodium hydrogen phosphate 11.3 g, potassiumdihydrogen phosphate 2.7 g, added to 1000 ml of distilled water) and the tubes were incubated at 37°C for 24 h with intermittent shaking. At the end of incubation period phosphate buffers as well as contents of the dialyzing bags were separately analysed for the concentration of ampicillin by spectrophotometric assay in triplicate. The extent of in vitro plasma protein binding of ampicillin was calculated by the following equation.

where,

CP’ = Concentration of drug in plasma after incubation

CB = Concentration of drug in buffer after incubation

CP = Concentration of drug in plasma before incubation

Binding capacity of the plasma protein to ampicillin (b_i) and the dissociation rate constant of protein drug complex (K_b) were calculated by the method of Pilloud (1973).

Ultraviolet-Visible-Spectrophotometer (Eppendorf™, USA) with 1 cm matched quartz cells was used for all spectral measurements. pH meter (Thermo scientific® Digital pH Meter Orion star A111), electronic weighing balance (A&D Instruments, India Pvt. Ltd.), refrigerated centrifuge (Eppendorf™, USA), hot water bath incubator shaker (Narang Scientific Works Pvt. Ltd), thoroughly clean and dried glasswares were used in the present study such as measuring cylinders, volumetric flasks and beakers were procured from Borosil Glass Works Ltd. (India). The glass wares were washed with hot water and detergent, rinsed with distilled water and finally dried in an oven prior to use.

Stock solution of 100 µg/ml ampicillin was prepared in distilled water. The standard solutions were prepared by dilution of stock solution with distilled water in a concentration range of 0.5, 1, 2, 4, 6, 8 and 10 gg/ml. The stock solution containing glass vials were wrapped in aluminium foil and stored under deep refrigeration (-20°C) until use.

Buffered standard solutions of ampicillin at a range of concentrations 0.5, 1, 2, 4, 6, 8 and 10 gg/ml were assayed by the specified procedure.

A simple and specific spectrophotometric method given by Smith et al. (1967) was applied for the determination of ampicillin at 320 nm, based on acid degradation product of penicillin at 75°C in the presence of citrate buffer (pH 5.2) and traces of copper salt. In the study, it was observed that this method has limitation, and the detection of ampicillin not beyond 1.0 gg/ml. We have now extended the method to the assay of ampicillin in horse plasma for in-vitro plasma protein binding assay.

The method was validated for linearity, precision (interday and intra-day), selectivity and sensitivity according to the ICH guidelines. Linearity could be established with standard plots constructed for ampicillin in the range of 1-10 µg.ml^-1 (Table 1). The experiment was repeated thrice on the same day and additionally on two consecutive days to determine intra-day and inter-day precision, respectively. Calibration curve was plotted between ampicillin concentrations against absorbance readings as shown in Fig. 1.

Plasma (0.2 ml) from heparinized blood is taken in a 1.5 ml centrifuge tube containing absolute ethanol (0.4 ml). After mixing in vortex for few seconds and centrifuging for 5 min at 4500 RPM, clear supernatant (0.2 ml) was taken into a 5 ml tube with citrate buffer solution (0.8 ml; pH 5.2) containing copper (15 µg/ml). The tube was agitated at 120 strokes/min, incubated at 75 °C for 30 min and then cooled in ice. The sample is taken in a 1.5 ml cuvette and read at 320 nm wavelength against the blank given by the residual non-incubated fraction of the supernatant.