Development, Validation and Application of an LC-MS/MS Bioanalytical Method for the Quantification of GF449, A Novel 5-HT1A Agonist, in Rat Plasma and Brain
We have recently reported a novel class of selective 5-HT1A agonists among which GF449 emerged for its high
potency and almost full agonist activity (pKi 5-HT1A = 8.8; pD2 = 9.22, %Emax = 91.6). In order to quantify GF449 in rat
plasma and brain, a sensitive LC-MS/MS method was developed and validated.
Solid phase extraction (SPE) or a combined protein precipitation SPE permitted an efficient analyte recovery and sample
clean-up. Multiple reaction monitoring (MRM) was used to track both GF449 and its internal standard (IS), MM189.
GF449 was determined and quantitated to nanomolar concentrations in both plasma and brain matrix (LOQs = 0.0025
nmol/mL). Specificity was ensured using three further MRM qualifier transitions for both analyte and IS. Linearity was
found in the range of 0.0025 nmol/mL to 1.00 nmol/mL (R2 = 0.9965) and from 0.0025 nmol/mL to 50 nmol/mL (R2 =
0.9999) for plasma and brain respectively. Intraday trueness ranged from 94.0% to 117.5% for brain and from 93.7% to
108.1% for plasma, while precision values were within 3.0% - 6.7% and 2.5% - 9.2% for plasma and brain respectively.
The interday trueness of plasma ranged from 89.6% to 107.7% and the precision values (CV%) ranged from 4.6% to
7.5%. Interday trueness and precision (CV%) of the brain ranged from 94.3% to 101.2% and from 1.6% to 11.5%
The method was validated in accordance with the EMEA guidelines and was successfully applied to plasma and brain
samples obtained from rats treated with a 10 mg/kg single oral dose of GF449, thus demonstrating its applicability to preclinical
Keywords: 5-HT1A receptors agonist, rat plasma, rat brain, LC-MS/MS, quantitative analysis, pharmacokinetics.
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