Alarelin Acetate

Alarelin, a gonadotropin‐releasing hormone analogue, is widely used in China for the treatment of endometriosis and uterine leiomyoma. In order to investigate its pharmacokinetic behavior and support the preclinical application of new formulations, we have developed a novel and highly selective bioanalytical method to determine alarelin in rat plasma based on liquid chromatography tandem mass spectrometry with triple stage fragmentation. After sample preparation by protein precipitation followed by reversed phase solid phase extraction, alarelin and triptorelin (internal standard) were chromatographed on an Ascentis® Express C18 column (50 mm × 4.6 mm, 2.7 µm) using gradient elution with 0.1% formic acid in water and acetonitrile at a flow rate of 1 mL/min. Detection was by positive mode electrospray ionization followed by triple stage fragmentation using the transitions at m/z 584.6→249.1→221.0 for alarelin and 656.5→249.1→176.0 for triptorelin, The assay was linear in the concentration range 0.3‐10 ng/mL with excellent precision and accuracy. It was successfully applied to a pharmacokinetic study in rats administered a dose of 13.5 µg/kg alarelin by intramuscular injection. The results show that the triple stage fragmentation strategy allows highly selective analysis of alarelin and has the potential to be widely applied to the bioassay of other peptidic drugs.

In the present study, for the first time electromembrane extraction followed by high performance liquid chromatography coupled with ultraviolet detection was optimized and validated for quantification of four gonadotropin‐releasing hormone agonist anticancer peptides (alarelin, leuprolide, buserelin and triptorelin) in biological and aqueous samples. The parameters influencing electromigration were investigated and optimized. The membrane consists 95% of 1‐octanol and 5% di‐(2‐ethylhexyl)‐phosphate immobilized in the pores of a hollow fiber. A 20 V electrical field was applied to make the analytes migrate from sample solution with pH 7.0, through the supported liquid membrane into an acidic acceptor solution with pH 1.0 which was located inside the lumen of hollow fiber. Extraction recoveries in the range of 49 and 71% within 15 min extraction time were obtained in different biological matrices which resulted in preconcentration factors in the range of 82–118 and satisfactory repeatability (7.1 < RSD% < 19.8). The method offers good linearity (2.0–1000 ng/mL) with estimation of regression coefficient higher than 0.998. The procedure allows very low detection and quantitation limits of 0.2 and 0.6 ng/mL, respectively. Finally, it was applied to determination and quantification of peptides in human plasma and wastewater samples and satisfactory results were yielded.