The aim of this study was to develop and to validate a UPLC-MS/MS method for the quantification of morphine, morphine-3-glucuronide, and morphine-6-glucuronide in mouse plasma and tissue homogenates to support preclinical pharmacokinetic studies.

The sample preparation consisted of protein precipitation with cold (2-8 °C) methanol:acetonitrile (1:1, v/v), evaporation of the supernatant to dryness, and reconstitution of the dry-extracts in 4 mM ammonium formate pH 3.5. Separation was achieved on a Waters UPLC HSS T3 column (150 x 2.1 mm, 1.8 µm) maintained at 50 °C and using gradient elution with a total runtime of 6.7 min. Mobile phase A consisted of 4 mM ammonium formate pH 3.5 and mobile phase B of 0.1% formic acid in methanol:acetonitrile (1:1, v/v). Detection was carried out by tandem mass spectrometry with electrospray ionization in the positive ion mode. The method was validated within a linear range of 1-2,000 ng/mL, 10-20,000 ng/mL, and 0.5-200 ng/mL for morphine, morphine-3-glucuronide, and morphine-6-glucuronide, respectively. In human plasma, the intra- and inter-run precision of all analytes, including the lower limit of quantification levels, were ≤ 15.8%, and the accuracies were between 88.1 and 111.9%. It has been shown that calibration standards prepared in control human plasma can be used for the quantification of the analytes in mouse plasma and tissue homogenates.

The applicability of the method was successfully demonstrated in a preclinical pharmacokinetic study in mice.

这项研究的目的是开发和验证一种UPLC-MS / MS方法，用于定量测定小鼠血浆和组织匀浆中的吗啡，吗啡-3-葡糖醛酸苷和吗啡-6-葡糖醛酸苷，以支持临床前药代动力学研究。

样品制备包括用冷（2-8°C）甲醇：乙腈（1：1，v / v）沉淀蛋白，将上清液蒸发至干，并在pH 3.5的4 mM甲酸铵中将干提取物重构。 。分离是在维持在50°C的Waters UPLC HSS T3色谱柱（150 x 2.1 mm，1.8 µm）上进行的，使用梯度洗脱的时间为6.7分钟。流动相A由4 mM甲酸铵pH 3.5和0.1％甲酸在甲醇：乙腈中的流动相B（1：1，v / v）组成。通过串联质谱在正离子模式下用电喷雾电离进行检测。对吗啡，吗啡-3-葡糖醛酸和吗啡-6-葡糖醛酸分别在1-2,000 ng / mL，10-20,000 ng / mL和0.5-200 ng / mL的线性范围内进行了验证。在人体血浆中 所有分析物的运行内和运行间精密度（包括定量下限）≤15.8％，准确度在88.1％至111.9％之间。已经显示，在对照人血浆中制备的校准标准品可用于定量小鼠血浆和组织匀浆中的分析物。

在小鼠的临床前药代动力学研究中成功证明了该方法的适用性。