Full length articleDevelopment and validation of a high-performance liquid chromatography-tandem mass spectrometry method to quantify LY‐354,740 in rat and marmoset plasma

- Development and validation of an ultra sensitive method LY-354,740 by UHPLC-HESI-MS/MS.
- The method uses a very limited volume of marmoset plasma for analysis.
- The chromatographic system uses a unique mode of separation (i.e. reverse-phase, cation-exchange and anion-exchange)..

LY-354,740 (eglumegad) is a selective and potent agonist of the metabotropic glutamate group II receptors (mGluR2,3) that has already entered clinical trials as a potential anti-psychotic agent and therefore has well-documented pharmacokinetic (PK), safety and tolerability profiles in human. Whereas its development as an anti-psychotic agent has not been pursued, LY-354,740 may have potential in other neuroscience-related fields, notably anxiety and neuro-protection. The common marmoset is a small primate that has long been used in neuroscience. However, given its small size and small circulating blood volume, conducting PK studies to determine the therapeutic effectiveness of LY-354,740 at clinically-relevant doses is challenging. Here, we have developed and validated a simple, sensitive and selective analytical method that enables quantification of LY-354,740 using a small volume of plasma. The analytical method consisted of protein precipitation followed by high-performance liquid chromatography with heat assisted electrospray ionisation mass spectrometry (UHPLC-HESI-MS/MS). The chromatographic separation was achieved using gradient elution with a mobile phase consisting of acetonitrile and 10 mM ammonium formate (pH 3) on a Thermo Scientific Acclaim Trinity P1 analytical column (100 x 3.0 mm I.D., 3 μm) operating at 45 °C and at a flow rate of 900 μl/min. The method displays a linear relationship ranging from 20.0 to 5000 ng/ml. Intra- and inter-day relative standard deviations are less than 5.7% and 7.0%, respectively and the accuracy ranged from 91.0 to 106.0%. The UHPLC-HESI-MS/MS analytical method we describe here is simple, sensitive, specific and capable of quantifying LY-354,740 in both rat and marmoset plasma, and is suitable to conduct PK studies after a single sub-cutaneous dose of 1.0 mg/kg or lower in both species.