Wang Ganfeng, Korfmacher Walter A
Department of Drug Metabolism and Pharmacokinetics, Schering-Plough Research Institute, 2015 Galloping Hill Road, Kenilworth, NJ 07033, USA.
Rapid Commun Mass Spectrom. 2009 Jul;23(13):2061-9. doi: 10.1002/rcm.4111.
This paper describes the development and partial validation of a fast, sensitive and specific ultra-performance liquid chromatography/tandem mass spectrometric method for the determination of 3-indoxyl sulfate (3-IS), an endogenous compound in mammals, in mouse plasma and brain samples. The analytical method involves direct dilution of samples with water and protein precipitation with acetonitrile containing an internal standard, followed by separation of 3-IS on a MonoChrom C(18) column and detected by selected reaction monitoring (SRM) in negative ionization mode using turbo ion-spray ionization. Due to high endogenous levels of 3-IS in control mouse plasma and brain, blank guinea pig plasma and brain were used for the preparation of standard curves and quality controls (QCs). The compound of interest was well separated from interference peaks from the matrices with a total runtime of 2.7 min under a gradient condition. The method was partially validated. The linear concentration range was 0.1 to 100 microg/mL in mouse plasma and 10 to 10,000 ng/g in mouse brain. Inter-assay mean bias and relative standard deviation (RSD) for plasma were in the range of -4.8% to 3.1% and 2.5% to 3.2%, respectively. Intra-assay mean bias and RSD for plasma were in the range of -3.3% to 1.4% and 1.9% to 2.8%, respectively. Inter-assay mean bias and RSD for brain were in the range of -1.8% to 3.5% and 1.7% to 8.1%, respectively. Intra-assay mean bias and RSD for brain were in the range of -1.7% to 3.9% and 4.1% to 7.3%, respectively. The lower limit of quantitation (LLOQ) for this assay was 0.1 microg/mL for plasma and 10 ng/g for brain. The matrix effect was not observed in both guinea pig plasma and mouse plasma.
本文描述了一种快速、灵敏且特异的超高效液相色谱/串联质谱法的开发及部分验证，该方法用于测定小鼠血浆和脑样本中的3-吲哚硫酸酯（3-IS），这是一种哺乳动物体内的内源性化合物。该分析方法包括用水直接稀释样本，并用含内标物的乙腈进行蛋白沉淀，随后在MonoChrom C(18)柱上分离3-IS，并使用涡轮离子喷雾电离在负离子模式下通过选择反应监测（SRM）进行检测。由于对照小鼠血浆和脑中3-IS的内源性水平较高，因此使用空白豚鼠血浆和脑来制备标准曲线和质量控制（QC）样本。在梯度条件下，目标化合物与基质中的干扰峰得到了很好的分离，总运行时间为2.7分钟。该方法经过了部分验证。在小鼠血浆中的线性浓度范围为0.1至100μg/mL，在小鼠脑中为10至10,000 ng/g。血浆的批间平均偏差和相对标准偏差（RSD）分别在-4.8%至3.1%和2.5%至3.2%的范围内。血浆的批内平均偏差和RSD分别在-3.3%至1.4%和1.9%至2.8%的范围内。脑的批间平均偏差和RSD分别在-1.8%至3.5%和1.7%至8.1%的范围内。脑的批内平均偏差和RSD分别在-1.7%至3.9%和4.1%至7.3%的范围内。该测定方法的血浆定量下限（LLOQ）为0.1μg/mL，脑为10 ng/g。在豚鼠血浆和小鼠血浆中均未观察到基质效应。
