Di Marco Giovana Seno, de Andrade Maria Claudina Camargo, Felipe Claudia Rosso, Alfieri Fernando, Gooding Ann, Silva Hélio Tedesco, Pestana José Osmar de Abrea, Casarini Dulce Elena
Department of Medicine, Nephrology Division, Federal University of São Paulo, Brazil.
Ther Drug Monit. 2003 Oct;25(5):558-64. doi: 10.1097/00007691-200310000-00004.
Different HPLC methods have been developed and used to determined sirolimus blood concentrations. These methods show different performance characteristics, mostly related to peak interference, recovery, assay sensitivity, and turnaround times.
We adapted, improved, and validated an HPLC method with UV detection for measurement of sirolimus in whole blood clinical samples.
The standards, quality controls, or patient samples (0.25 or 0.5 mL) and internal standard (desmethoxysirolimus) were extracted with 1-chlorobutane. After evaporation, the extract was reconstituted in a 70% acetonitrile/water mixture and analyzed onto a reverse-phase C18 column at 50 degrees C under a flow rate of 1.0 mL/min in the HPLC system. Ultraviolet detection was performed at 278 nm, with sensitivity setting of 0.010 AUFS. Identification of peaks of interest was by retention time; quantification of sirolimus was based on a peak area ratio.
Analytic recovery ranging from 96 to 120% (CV = 3.7 to 16.8%; bias = -4.2 to 16.7%) was observed throughout the assay's linear range (2.5-150.0 ng/mL). The lower limit of quantification for both sample volumes (0.25 or 0.5 mL) was 2.5 ng/mL (CV = 12 and 15%, bias = -1.2 and 4%, respectively). The intra- and interassay imprecision ranged from 6.2 to 14.4% and from 9.1 to 18.6%, with bias ranging from 1.3 to 12.9% and -1.8% to 7.1, for quality control levels of 3, 10, and 20 ng/mL. Whole blood and extracted samples are stable at room temperature and at 4 and -20 degrees C for 1 week and 3 days, respectively. Chromatograms showed good separation free of interfering peaks. A set of 45 samples can be extracted in 2 h, allowing results within 24 h.
This HPLC-UV method shows good and reproducible performance, satisfying all requirements of an assay designated to be applied in therapeutic drug monitoring strategies after organ transplantation.
已开发并使用不同的高效液相色谱（HPLC）方法来测定西罗莫司的血药浓度。这些方法表现出不同的性能特征，主要与峰干扰、回收率、分析灵敏度和周转时间有关。
我们对一种采用紫外检测的HPLC方法进行了调整、改进和验证，用于测定全血临床样本中的西罗莫司。
将标准品、质量控制品或患者样本（0.25或0.5 mL）以及内标（去甲氧基西罗莫司）用1 - 氯丁烷萃取。蒸发后，提取物用70%乙腈/水混合物复溶，并在HPLC系统中于50℃下以1.0 mL/min的流速在反相C18柱上进行分析。在278 nm处进行紫外检测，灵敏度设置为0.010 AUFS。通过保留时间鉴定感兴趣的峰；西罗莫司的定量基于峰面积比。
在整个分析线性范围（2.5 - 150.0 ng/mL）内，分析回收率在96%至120%之间（CV = 3.7%至16.8%；偏差 = -4.2%至16.7%）。两种样本体积（0.25或0.5 mL）的定量下限均为2.5 ng/mL（CV分别为12%和15%，偏差分别为 -1.2%和4%）。对于3、10和20 ng/mL的质量控制水平，批内和批间不精密度范围分别为6.2%至14.4%和9.1%至18.6%，偏差范围分别为1.3%至12.9%和 -1.8%至7.1%。全血和萃取后的样本分别在室温、4℃和 -20℃下稳定1周和3天。色谱图显示分离良好，无干扰峰。一组45个样本可在2小时内萃取完毕，24小时内出结果。
这种HPLC - UV方法表现出良好且可重复的性能，满足器官移植后治疗药物监测策略中指定分析方法的所有要求。
