An assay using HPLC with fluorescence (FL) detection method for monitoring native FL of tocilizumab (TCZ) in human serum combined with extremely simple and rapid pretreatment without any antigen-antibody reaction was developed. Good separation of TCZ was achieved within 13 min on a Presto FF-C18 column (100 × 4.6 mm i.d., 2 µm). Simple pretreatment with acetonitrile containing primary and secondary alkylamines having longer than C3 in the alkyl chain removed immunoglobulin G subclass 1 and TCZ could be recovered selectively. The spiked calibration curve of TCZ in human serum showed good linearity in the range of 40–1000 µg/mL (r > 0.997). The lower limit of quantitation (S/N = 10) of the TCZ was 19.7 µg/mL. The accuracy was within 103.5–114.9%, and the intra- and inter-day precisions as relative standard deviations were less than 5.3 and 7.8% (n = 5), respectively. The recovery of TCZ was 42.2 ± 3.4% (n = 3). The TCZ in pretreated sample was confirmed to be stable for 6 h (>95%) at room temperature and 24 h (>95%) at 4 °C. The proposed method is considered extremely superior to the previous methods in terms of time requirement for analysis. Therefore, the developed method may be more useful than conventional methods in urgent situations, such as confirming therapeutic efficacy of cytokine-release syndrome by 2019 coronavirus disease.

开发了一种使用 HPLC 和荧光 (FL) 检测方法监测人血清中托珠单抗 (TCZ) 的天然 FL，并结合极其简单和快速的预处理，没有任何抗原抗体反应。使用 Presto FF-C18 色谱柱（100 × 4.6 mm 内径，2 µm）在 13 分钟内实现了 TCZ 的良好分离。用含有烷基链中长于 C3 的伯和仲烷基胺的乙腈进行简单预处理，可以选择性地回收免疫球蛋白 G 亚类 1 和 TCZ。人血清中 TCZ 的加标校准曲线在 40–1000 µg/mL 范围内显示出良好的线性 ( r  > 0.997)。定量下限（S/ N = 10) 的 TCZ 为 19.7 µg/mL。准确度在 103.5–114.9% 之间，作为相对标准偏差的日内和日间精密度分别小于 5.3 和 7.8% ( n  = 5)。TCZ 的回收率为 42.2 ± 3.4% ( n  = 3)。经证实，预处理样品中的 TCZ 在室温下稳定 6 小时 (>95%)，在 4°C 下稳定 24 小时 (>95%)。就分析的时间要求而言，所提出的方法被认为极其优于以前的方法。因此，所开发的方法在紧急情况下可能比常规方法更有用，例如通过 2019 冠状病毒病确认细胞因子释放综合征的治疗效果。