TITLE: Pharmacokinetics and bioequivalence of two imidocarb formulations in cattle after subcutaneous injection
AUTHORS: Anonymous  , Chen Chen, Maolin Liu, Xiaojie Chen, Chunshuang Liu, Yanyan Feng, Xinbo Yan, Yiming Liu, Xiubo Li, Honglei Wang

[DESCRIPTION]
Imidocarb (IMD) is commonly used for treatment of eperythrozoon, babesia, piroplasma and trypanosoma in animals, but there are few studies on its pharmacokinetics in cattle. The purpose of this study was to obtain pharmacokinetic parameters and assess the bioequivalence of subcutaneous injections of two IMD formulations in cattle. Forty-eight healthy cattle, 24 males and 24 females, were randomly divided into two groups (test group and reference group) with 12 males and 12 females per group. The generic IMD was injected subcutaneously with a single dose of 3.0 mg/kg in the test group. Reference group animals were given one injection of the marketedIMDat the same dosage. The limit of detection (LOD) and limit of quantification (LOQ) for IMD in cattle plasma were 0.05 μg/Land 0.1 μg/L, respectively. The recoveries ranged from 88.50% to 92.42%, and the equation of this calibration curve was Y=13672.1X+187.43. The pharmacokinetics parameters of the test group showed that the maximum concentration of 2257.5±273.62 µg/L was obtained at 2.14±0.67 h, AUC0-t 14553.95±1946.85 µg·h/L, AUC∞15077.88±1952.19 µg·h/L, T1/2 31.77±25.75 h, CL/F 0.14±0.02 L/h/kg, and Vz/F 6.53±5.34 L/kg. There was no significant difference in AUC0-t, AUC∞ and Cmax between the test group and the reference group (P＞0.05). The 90% confidence interval of AUC0-t,AUC0-∞ andCmax in the test group was included in 80%–125% AUC0-t, AUC0-∞and 70%–143% Cmax in the reference group, respectively. Based on these results, the two preparations were found to be bioequivalent. 

[STEPS]
1. Sample collection 
Blood samples (10–15 mL) were collected from the jugular vein at 0 h before administration and 10 min, 30 min, 1, 2, 4, 6, 8, 10, 12, 24, 48, 72 and 96 h after subcutaneous administration, and were drawn in vacutainers containing disodium EDTA as anticoagulant. The samples were immediately centrifuged at 1500 g for 10 min. All the plasma samples in plastic vials were stored at −20°C until they were analyzed.
2. Sample preparation
We used a weak cation-exchange solid phase extraction procedure described by Tarbin [10], and made some modifications to determine the amount of IMD in the plasma. Brieﬂy, 1.0 mL plasma samples were added to plastic centrifuge tubes, adding 3 mL methanol/acetonitrile (90/10, v/v), vertexing for 1 min, ultrasonicating for 15 min, and centrifuging for 10 min at 7000 g. To improve IMD recoveries, the residues were extracted twice. The second extracted solution was combined with the first. The combined extracted solution was passed through SPE columns (Waters Oasis WCX, 3cc 60 mg, Waters Company, USA) conditioned with 3 mL methanol and 3 mL water. The loaded cartridge was washed with 3 mL methanol/water (50/50, v/v), and was eluted with 3 mL methanol/formic acid (96/4, v/v). The analyte was evaporated under a nitrogen stream at 40°C, reconstituted in 1 mL methanol/water (15/85, v/v), and filtered through a 0.22 µm nylon syringe filter before analysis by UPLC-MS/MS.