The objective of this study was to develop and validate a method using liquid chromatography coupled with a mass spectrometer (LC–MS/MS) for simultaneous analysis of adenosine triphosphate (ATP), adenosine diphosphate (ADP) and adenosine monophosphate (AMP) levels in dry blood spot (DBS), liver, brain (parietal and occipital regions) of BALB/c neonatal mice pups, using ¹³C₁₀ ATP as internal standard (IS). Simultaneous nucleotides separation of the investigated samples was achieved in 5 min on a C18 column [Waters × Bridge C18, (4.6 × 50 mm, 3.5 μm)] using a gradient elution of 4 mM ammonium acetate and 100% acetonitrile as mobile phase A and mobile phase B, respectively. MS detection was performed in an ESI positive ionization mode. The method was validated according to the international council for harmonization guidelines. The standard calibration curve was linear over the concentration range of 0.1, 1, 5, 10, 15, 20 µg/ml of standard mixtures with known quantity of internal standards (IS) and the data showed good linearity (R² 0.999), with lower limit of quantification (LLOQ 0.1 µg/ml). The within run and between run precision of QCs (%CV) were ≤ 15% and accuracy (%) of measured concentration of QCs ranged between 88 and 110%. All the processed QCs were stable after applying the various storage conditions. The robustness study showed no significant variance in analyte peak quality and quantity with the smaller variation of temperature, pH and acetonitrile concentration. The matrix factor was in acceptable range. The present validation results show that the developed LC–MS/MS method is suitable to evaluate ATP, ADP and AMP levels in DBS, liver and brain tissues of neonatal mice pups.

本研究的目的是开发和验证一种使用液相色谱与质谱联用 (LC-MS/MS) 同时分析三磷酸腺苷 (ATP)、二磷酸腺苷 (ADP) 和一磷酸腺苷 (AMP) 水平的方法。 BALB/c 新生小鼠幼崽的干血斑 (DBS)、肝脏、大脑（顶叶和枕叶区域），使用¹³ C ₁₀ATP 作为内标 (IS)。在 C18 柱 [Waters × Bridge C18, (4.6 × 50 mm, 3.5 μm)] 上使用 4 mM 醋酸铵和 100% 乙腈作为流动相 A 和流动相 B。MS 检测在 ESI 正电离模式下进行。该方法根据国际协调委员会指南进行了验证。标准校准曲线在 0.1、1、5、10、15、20 µg/ml 与已知内标量 (IS) 的标准混合物的浓度范围内呈线性，数据显示出良好的线性 (R ²0.999），定量下限（LLOQ 0.1 µg/ml）。QC 的运行内和运行间精密度 (%CV) ≤ 15%，QC 测量浓度的准确度 (%) 介于 88% 和 110% 之间。在应用各种储存条件后，所有处理过的 QC 都是稳定的。稳健性研究表明，随着温度、pH 和乙腈浓度的变化较小，分析物峰的质量和数量没有显着变化。矩阵因子在可接受的范围内。目前的验证结果表明，开发的 LC-MS/MS 方法适用于评估新生小鼠幼崽 DBS、肝脏和脑组织中的 ATP、ADP 和 AMP 水平。