Zhao Yue-Tao, Dai Hao-Ran, Li Yue, Zhang Yuan-Yuan, Guo Hong-Li, Ding Xuan-Sheng, Hu Ya-Hui, Chen Feng
Pharmaceutical Sciences Research Center, Department of Pharmacy, Children's Hospital of Nanjing Medical University, Nanjing, China.
School of Basic Medicine and Clinical Pharmacy, China Pharmaceutical University, Nanjing, China.
Front Pharmacol. 2022 Sep 6;13:925018. doi: 10.3389/fphar.2022.925018. eCollection 2022.
Sirolimus (SRL) is a mammalian target of rapamycin (mTOR) inhibitor. The whole blood concentration of SRL is routinely monitored to tailor dosage and prevent toxicity. Currently, the enzyme multiplied immunoassay technique (EMIT) is often applied to perform therapeutic drug monitoring (TDM) of SRL, but the cross-reactivity with various metabolites is of great concern. A more specific method is required, such as liquid chromatography-tandem mass spectrometry (LC-MS/MS). However, no study on the method comparison of the EMIT and LC-MS/MS for the measurement of whole blood SRL concentration in children with vascular anomalies has been reported. This study developed a simple and sensitive LC-MS/MS assay for the determination of SRL. Meanwhile, consistency between LC-MS/MS and the EMIT was evaluated by linear regression and Bland-Altman analysis. Whole blood samples were deproteinized with methanol for erythrocyte lysis, and the resulting solution was injected into the LC-MS/MS system using the positive electrospray ionization mode. The multiple reaction monitoring transitions of 931.7 → 864.6 and 934.7 → 864.6 were used for SRL and SRL-d as the internal standards, respectively. The analytes were separated on a C18 column with a gradient mobile phase (0.1 mM formic acid and 0.05 mM ammonium acetate in methanol/ultrapure water). Blood samples collected from children with vascular anomalies undergoing SRL therapy were tested by EMIT and by LC-MS/MS. The linear range of LC-MS/MS was 0.500-50.0 ng/ml and that of the EMIT was 3.50-30.0 ng/ml. A significant positive correlation between the two assays was established with a regression equation described as [ ] = 1.281 × [ ] + 2.450 ( = 0.8361). Bland-Altman plots showed a mean concentration overestimation of 4.7 ng/ml [95% CI: (-3.1, 12.6)] and a positive bias of 63.1% [95% CI: (-36.1, 162.3)] generated by the EMIT more than that of by LC-MS/MS. In conclusion, the two methods were closely correlated, indicating that switching between the two methods is feasible. Considering the overestimation nature of the EMIT assay, switching from the EMIT to the LC-MS/MS method deserves close attention and necessary re-evaluation for the target therapeutic reference range, may be required when methods are switched within the same clinical laboratory or results are compared between different laboratories.
西罗莫司（SRL）是一种雷帕霉素哺乳动物靶点（mTOR）抑制剂。常规监测SRL的全血浓度以调整剂量并预防毒性。目前，酶倍增免疫分析技术（EMIT）常被用于SRL的治疗药物监测（TDM），但其与各种代谢物的交叉反应性备受关注。需要一种更特异的方法，如液相色谱 - 串联质谱法（LC - MS/MS）。然而，尚未有关于在血管异常儿童中测量全血SRL浓度时EMIT与LC - MS/MS方法比较的研究报道。本研究开发了一种简单且灵敏的LC - MS/MS法用于测定SRL。同时，通过线性回归和Bland - Altman分析评估了LC - MS/MS与EMIT之间的一致性。全血样本用甲醇进行脱蛋白以裂解红细胞，所得溶液采用正电喷雾电离模式注入LC - MS/MS系统。分别以931.7→864.6和934.7→864.6的多反应监测跃迁用于SRL和SRL - d作为内标。分析物在C18柱上用梯度流动相（甲醇/超纯水中的0.1 mM甲酸和0.05 mM乙酸铵）进行分离。对接受SRL治疗的血管异常儿童采集的血样进行EMIT和LC - MS/MS检测。LC - MS/MS的线性范围为0.500 - 50.0 ng/ml，EMIT的线性范围为3.50 - 30.0 ng/ml。两种检测方法之间建立了显著的正相关，回归方程为[ ] = 1.281×[ ] + 2.450（ = 0.8361）。Bland - Altman图显示，EMIT产生的平均浓度高估为4.7 ng/ml [95%可信区间：（-3.1, 12.6）]，正偏差为63.1% [95%可信区间：（-36.1, 162.3）]，高于LC - MS/MS。总之，两种方法密切相关，表明在两种方法之间切换是可行的。考虑到EMIT检测的高估性质，从EMIT切换到LC - MS/MS方法值得密切关注，并且当在同一临床实验室切换方法或在不同实验室比较结果时，可能需要对目标治疗参考范围进行必要的重新评估。
