A rapid, highly sensitive and simple high-performance liquid chromatographic–tandem mass spectrometric (LC–MS) assay is developed and validated for the quantification of leuprolide: a Gonadotropin Releasing Hormone analog (GnRH) in human plasma. Moreover, various parameters of the method stability are determined. After the addition of stable isotope (internal standard), the leuprolide was extracted from human plasma by a C₁₈ solid phase micro extraction (MEPS) cartridge and directly injected into LC-MS/MS system. Chromatographic separation was achieved using a Waters Atlantis HILIC, C₁₈, 150 × 2.1 mm, 5 μ column. Mobile phase was a mixture of acetate buffer (pH 3) and acetonitrile (25/75). Drug detection was performed by MS using electrospray ionization in positive mode. Multiple reaction monitoring (MRM) with a tandem mass spectrometer was used to detect the analytes. Precursor to product ion transitions of: m/z 605.5 → m/z 110.2 and m/z 609.1 → m/z 249.1 were used to quantify leuprolide and leuprolide-¹³C₆-¹⁵N, respectively. Sample analysis time was 3 min for each injection. The assay exhibited a linear dynamic range of 0.0500–40 ng/ml for each analyte with a lower limit of quantification (LLOQ) of: 0.0500 ng/ml. Furthermore, a complete analytic validation was carried out, including tests on: The specificity, precision, accuracy, matrix effect and stability under different storage conditions. Importantly, the obtained results established: an acceptable precision and accuracy for concentration over standard curve range. Nevertheless, it is to emphasize the simplicity, rapidity and also the high precision and accuracy of this novel LC–MS method, offering useful information about solution stability. Finally, this work is a good alternative to quantify Leuprolide concentration in human blood, especially on human clinic trials step.

开发了一种快速，高度灵敏，简单的高效液相色谱-串联质谱（LC-MS）测定法，并验证了亮丙瑞林的定量：人血浆中的促性腺激素释放激素类似物（GnRH）。此外，确定了方法稳定性的各种参数。添加稳定同位素（内标）后，通过C ₁₈固相微萃取（MEPS）柱从人血浆中提取亮丙瑞林，然后直接注入LC-MS / MS系统。使用Waters Atlantis HILIC，C ₁₈色谱分离，150×2.1 mm，5μ色谱柱 流动相是乙酸盐缓冲液（pH 3）和乙腈（25/75）的混合物。使用正离子模式下的电喷雾电离通过MS进行药物检测。使用具有串联质谱仪的多反应监测（MRM）来检测分析物。前体的产物离子跃迁：米/ ž 605.5→ 米/ Ž 110.2和米/ ž 609.1→ 米/ ž 249.1被用来量化亮丙瑞林和leuprolide- ¹³ Ç ₆ - ¹⁵N分别。每次进样的样品分析时间为3分钟。该分析法显示每种分析物的线性动态范围为0.0500–40 ng / ml，定量下限（LLOQ）为：0.0500 ng / ml。此外，还进行了完整的分析验证，包括以下方面的测试：特异性，精密度，准确性，基质效应和在不同储存条件下的稳定性。重要的是，确定了获得的结果：在标准曲线范围内浓度的可接受的精度和准确度。尽管如此，它还是要强调这种新颖的LC-MS方法的简便性，快速性以及高精度和高精度，并提供有关溶液稳定性的有用信息。最后，这项工作是定量人体血液中亮丙瑞林浓度的好选择，尤其是在人类临床试验步骤中。