Previous studies using immunohisoflurescence or radioimmunoassay had demonstrated to evaluate the concentration of buserelin in plasma However, antibody preparation is included in this method, which is very time consuming [7, 16, 17]. Reverse phase high-performance liquid chromatography (RP-HPLC) was used for analysis of gonadorelin analogues. but an ion pair agent was need which trifluoracetic acid created a low pH environment [18, 19]. Capillary electrophoresis (CE), a powerful tool, was used to analyze the peptide. However, a high ionic strength of buffer was selected to decrease absorption of buserelin in the analysis [20-22]. Several technical tools were combined with CE to detect the concentration. These methods can speed up the quantification of buserelin, but pH 3.0 was needed for successful separation of the solution[22, 23]. The HPLC method reduces the retention time from 40 min to 9.2 min [24]. Currently, UPLC-MS/MS is used to detect peptides and proteins for doping control. The sample preparation was dilute-shoot (DS) or solid-phase extraction (SPE), which will be cost-effectiveness and loss of sensitivity [25]. In our research, a rapid and sensitive UPLC-MS-MS method was established and validated following FDA guidance to evaluate busesrelin levels in blood samples.
In this study, tmax was achieved after 0.57 ± 0.18 h for pigs and 1.05 ± 0.27 h for cows which is longer than reported buserelin solution in rats (45 min) at a single dose of 6 mg/kg following s.c. administration [13], health volunteers (20 min, 42 min, 58 min, 43.8 min, 38.8 min) after being administered at a single dose of 500 µg i.v., 5 µg s.c., 150 µg i.n., 300 µg i.n., and 450 µg i.n., respectively [3], and shorter than buserelin suspension in rats (180 min) at 6 mg/kg dose following s.c. administration, rats (1.92 ± 0.42 h) at a single dose of 0.1 mg/kg i.n., and dogs (4 h) after being administered s.c. at a dose of 3.3 mg [17]. The tmax reflects the rate of absorption, which indicated that buserelin in pigs was absorbed very quickly due to the rate of metabolic rate of organs to the whole body [26].These differences in the parameters show that buserelin is absorbed at a faster rate in pigs than in cows. These data agree with the view that small animals eliminate the drugs more rapidly than large ones. However, compared the data of health volunteers with the one of rats following i.n., the conclusion seems paradox. It can be explained by the fact that rats using in the experiment were anesthetized. The physical condition can affect absorption progress.
The Vd was 80.49 ± 43.88 L for pigs and 839.88 ± 174.77 L for cows, which is larger than pigs (304 ± 112 mL/kg) at a single dose 1 mg i.v.[14], and dogs (50.1 ± 2.4 mL/kg) at a single dose 5 mg i.v.[17]. As it means that the ratio of the whole amount of drug to the plasma concentration, our results showed that animals using in this study had larger volume of body water or extracellular water[26]. High plasma concentration, high bonding rate, means more drugs cannot across the membrane and barrier. Therefore, binding changes can affect the distribution of drugs. As protein binding is about 15% [2], it is proportional to the body volume and body weight [26]. It has been reported that buserelin is rapidly degraded by pyroglutamyl-amino-peptidase which can be isolated from mammalian liver [2].The main serum metabolite was buserelin (5-9) pentapeptide[3]. Its intact form and metabolites are mainly excreted through urine [14, 26]. This view has been proved correct when compared with the clearance of buserelin solution in rats (30.34 ± 2.12 mL/min) at 6 mg/kg s.c. injection [13], in dogs (1.7 ± 0.10 mL/kg/min) at 5 mg per dog i.v. injection [17], and in pigs (2.0 ± 0.4 mL/kg/min) at 1 mg per pig i.v. injection These data show that hepatic blood flow is the major determinants for the elimination process because of it has an allometric relationship with body weight [26]. The elimination half-time is proportional to its volume of distribution, but inversely to its clearance. The t1/2 value was in pigs 1.29 ± 0.40 h and in cows 1.13 ± 0.30 h which is longer than in rats (5 min) in guinea pigs (12 min) following i.v. application [1], in rats (42 min) at a single dose of 6 mg/kg s.c. injection[13] and in dogs (56.4 ± 0.98 min) at a single dose of 5 mg i.v. injection[17], approximately equate to the value in pigs (103 ± 20 min) at a single dose of 1 mg i.v.injection [14]. However, the elimination half-time of buserelin in human has a 72-120 min regardless of the administration route [2, 3]. These data show that pigs and cows have a low elimination process which can be related with the rate of metabolism.
Similar to other peptide hormones, buserelin after oral administration will be largely digested. The pharmacokinetic data obtained following oral administration showed a short half-life and a rapid clearance due to degradation into smaller metabolites without biological activity. However, there are some attempts which can slow-down absorption from delivery site or slow-down enzymatic degradation and elimination to improve the bioavailability, such as sodium glycodeoxycholate, Zn2+ suspension within the buserelin solution, and cyclodextrin derivatives[13, 14, 16]. Meanwhile some reports hold the view that buserelin administration induces loss of erectile potency, hot flush[9], uterine bleeding[6], apoptosis in spermatozoa lineage and inhibits immune system function [27, 28], further investigations are required to assess its side effects in practical applications.