Franchini Silvia, Taddia Laura, Pinetti Diego, Carnevale Gianluca, Brasili Livio
Dipartimento di Scienze Farmaceutiche, Università degli Studi di Modena e Reggio Emilia, Via Campi 183, 41100 Modena, Italy.
Med Chem. 2014;10(5):449-59. doi: 10.2174/1573406409666131128150507.
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 (R(2) = 0.9965) and from 0.0025 nmol/mL to 50 nmol/mL (R(2) = 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% respectively. 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 pharmacokinetic studies.
我们最近报道了一类新型的选择性5-HT1A激动剂，其中GF449因其高效能和几乎完全的激动剂活性而脱颖而出（5-HT1A的pKi = 8.8；pD2 = 9.22，最大效应百分比 = 91.6）。为了定量大鼠血浆和脑中的GF449，开发并验证了一种灵敏的液相色谱-串联质谱（LC-MS/MS）方法。固相萃取（SPE）或联合蛋白沉淀的SPE实现了有效的分析物回收和样品净化。采用多反应监测（MRM）来追踪GF449及其内标（IS）MM189。在血浆和脑基质中均测定并定量出GF449的纳摩尔浓度（定量下限 = 0.0025 nmol/mL）。通过对分析物和内标使用另外三个MRM定性离子对来确保特异性。血浆和脑中的线性范围分别为0.0025 nmol/mL至1.00 nmol/mL（R(2) = 0.9965）和0.0025 nmol/mL至50 nmol/mL（R(2) = 0.9999）。脑内日间准确性范围为94.0%至117.5%，血浆为93.7%至108.1%，而血浆和脑的精密度值分别在3.0% - 6.7%和2.5% - 9.2%范围内。血浆的日间准确性范围为89.6%至107.7%，精密度值（CV%）范围为4.6%至7.5%。脑的日间准确性和精密度（CV%）分别为94.3%至101.2%和1.6%至11.5%。该方法按照欧洲药品评价局（EMEA）指南进行了验证，并成功应用于经10 mg/kg单剂量口服GF449处理的大鼠的血浆和脑样品，从而证明了其在临床前药代动力学研究中的适用性。
