Title : Analytical method development and validation of End-LAA for in vitro and in vivo studies
Abstract:
Endomorphin-1 is more effective in the treatment of neuropathic pain than morphine-like analgesics and side effects after treatment are less due to its endogenous nature. However, endomorphin-1 cannot be clinically used for reasons such as short duration of action, poor metabolic stability, vaginal bleeding, and failure to pass through the blood-brain barrier. Therefore lipid-modified derivate of endomorphin-1 peptide (End-LAA) was prepared to provide its oral bioavailability. Current study comprises the analytical method development and validation to determine End-LAA amount in in vitro studies and in vivo biological samples.
Liquid chromatography–mass/mass spectrometry (LC-MS/MS) method was developed using ACE C18 (50mm x 4.6 mm x 5µm) column with a flow rate of 0.5 mL/min. End-LAA level measurement was performed by electrospray ionization (ESI) method and positive ion mode using an Agilent 6420 LC-MS / MS instrument. The mobile phase was consisted of 0.1% v/v formic acid solution in acetonitrile and 0.1% v/v formic acid solution in water. The main mass/charge (m/z) value of End-LAA was 780.3, and the Q3 m/z values obtained therefrom were 448.3 and 284.1. The fragmentor voltage was applied as 160 V. Standard solutions of End-LAA in acetonitrile and plasma were prepared within a concentration range of 0.19- 100 ng/mL. The calibration curves were obtained with R2 values of 0.995 -0.998. The proposed method was extensively validated in terms of precision, accuracy, linearity and range. Besides, the limit of detection and quantification values were also calculated and discussed according to Guidelines.
As a result a simple, selective and rapid LC–MS/MS method has been developed and validated for the sensitive determination of End-LAA.
The authors wish to thank Prof. Dr Istvan Toth (University of Queensland, Australlia) for synthesis and providing of EndLAA.
