A quick and sensitive liquid chromatography-mass spectrometry technique was designed, improved, and validated for simultaneous determination of Empagliflozin (EPG) and Linagliptin (LNG) using Empagliflozin-d4 (EPG-d4) and linagliptin-d4 (LNG-d4) as internal standards (IS) in rat plasma. Target analytes and the IS were extracted using freezing lipid precipitation (FLP) and optimized using the strong cation exchange solid phase extraction (SCX-SPE) method to achieve the maximum sample clean-up. In particular, when combined with SPE clean-up, FLP can efficiently eliminate the plasma sample's high lipid content. More than 84.14% of plasma lipids were rapidly removed during the FLP procedure, with minimal loss of EPG and LNG. We used LC-atmospheric chemical ionization (APCI)-mass spectrometry was employed to assess the efficiency of FLP in lipid removal. The SCX-SPE cartridges removed the remaining impurities from EPG and LNG, allowing for further purification. The samples were chromatographically separated using a Spherisorb RP/Cyano column by pumping a gradient mobile phase comprised of acetonitrile and 25 mM ammonium acetate buffer (pH 8.1) in positive ion mode at a flow rate of 0.8 mL/min. The selected reaction monitoring technique was performed using a Waters triple-stage quadrupole tandem mass spectrometer equipped with an electrospray ionization (ESI) source. The chromatographic separation was accomplished using a Waters Acquity® high-performance liquid chromatography (HPLC) system. Mass transition (m/z) of 451.15/71.12 for EPG, m/z 473.27/419.94 for LNG; m/z 455.19/71.12 for EPG-d4, and 477.27/423.94 for LNG-d4 was successfully achieved. This study successfully examined the concentration ranges of 25-1050 ng/mL for EPG and 0.35-15 ng/mL for LNG. The results showed that the linearity of EPG ranged from 25.14 to 985.26 ng/mL, while the linearity of LNG ranged from 0.59 to 14.86 ng/mL. The relative standard deviation (RSD) for both EPG and LNG, within and between days, were below 3.83%, indicating that they fall within acceptable limits. This novel approach demonstrated favourable outcomes in a pharmacokinetic study involving healthy rats, where EPG and LNG were co-administered. This study found that the co-administration of both drugs did not have a significant impact on their pharmacokinetic behavior, suggesting the absence of any drug-drug interactions.

中文翻译：

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使用冷冻脂质沉淀和 SCX-SPE 辅助 HPLC-MS/MS 方法开发和验证大鼠血浆中恩格列净和利格列汀的同时测定及其在药代动力学研究中的应用。

设计、改进和验证了一种快速灵敏的液相色谱-质谱技术，可使用恩格列净-d4 (EPG-d4) 和利格列汀-d4 (LNG-d4) 作为内部同时测定恩格列净 (EPG) 和利格列汀 (LNG)。大鼠血浆中的标准品（IS）。使用冷冻脂质沉淀 (FLP) 提取目标分析物和内标，并使用强阳离子交换固相萃取 (SCX-SPE) 方法进行优化，以实现最大程度的样品净化。特别是，当与 SPE 净化相结合时，FLP 可以有效消除血浆样品中的高脂质含量。在 FLP 过程中，超过 84.14% 的血浆脂质被快速去除，EPG 和 LNG 的损失最小。我们使用 LC-大气化学电离 (APCI)-质谱法来评估 FLP 去除脂质的效率。SCX-SPE 柱去除了 EPG 和 LNG 中的剩余杂质，以便进一步纯化。使用 Spherisorb RP/Cyano 柱，通过以 0.8 mL/min 的流速泵送由乙腈和 25 mM 醋酸铵缓冲液 (pH 8.1) 组成的梯度流动相，以正离子模式对样品进行色谱分离。所选择的反应监测技术是使用配备电喷雾电离（ESI）源的沃特世三级四极杆串联质谱仪进行的。使用 Waters Acquity® 高效液相色谱 (HPLC) 系统完成色谱分离。EPG 的质量转变 (m/z) 为 451.15/71.12，LNG 的质量转变 (m/z) 为 473.27/419.94；EPG-d4 的 m/z 为 455.19/71.12，LNG-d4 的 m/z 为 477.27/423.94。本研究成功检验了 EPG 的浓度范围为 25-1050 ng/mL，LNG 的浓度范围为 0.35-15 ng/mL。结果表明，EPG的线性范围为25.14~985.26 ng/mL，LNG的线性范围为0.59~14.86 ng/mL。EPG 和 LNG 在日内和日间的相对标准偏差 (RSD) 均低于 3.83%，表明它们处于可接受的限度内。这种新颖的方法在一项涉及健康大鼠的药代动力学研究中证明了良好的结果，其中 EPG 和 LNG 共同给药。这项研究发现，两种药物的共同给药对其药代动力学行为没有显着影响，表明不存在任何药物间相互作用。