采用液相色谱-同位素稀释质谱法测定全血中免疫抑制药物的方法

Procedure for determination of immunosuppressive drugs in whole blood with liquid chromatography-isotope dilution mass spectrometry.

作者信息

Molnár Petra Magdolna, Dux László, Reinauer Hans, Kress Michael, Akerboom Theodorus, Szederkényi Edit, Kaiser Patricia

机构信息

Department of Biochemistry, Faculty of Medicine, University of Szeged, Szeged, Hungary.

出版信息

Clin Lab. 2011;57(11-12):983-92.

PMID:22239031

Abstract

BACKGROUND

Cyclosporin A, sirolimus, tacrolimus, and everolimus are immunosuppressive drugs used for therapy after organ transplantation. There are several analytical procedures for monitoring the drug level in blood, e.g. immunological methods and high-performance liquid chromatography combined with mass spectrometry (MS). From external quality assessment schemes, it became evident that the analytical results show high dispersion and further standardization is required.

METHODS

Liquid/liquid extraction of the drugs from whole blood samples was performed using ammonium acetate buffer, pH 9.5, and tert-butylmethyl ether/ethyl acetate (1:1 v/v). Separation of the immunosuppressive drugs was achieved by HPLC using a phenyl-hexyl-RP column with a ternary gradient elution profile, consisting of water, methanol, and acetonitrile containing 0.1% v/v formic acid and 0.1 mmol/L Cs+. Quantification of immunosuppressive drugs was performed by isotope-dilution mass spectrometry using [2H12]-Cyclosporin A [13C, 2H3]-Rapamycin, [13C, 2H2]-Tacrolimus, and [13C2, 2H4]-42-O-(2-Hydroxyethyl)rapamycin as internal standards.

RESULTS

The recovery of the new procedure was determined by analysis of spiked blood samples. The recovery in spiked EDTA whole blood samples was 100.8 - 102.5% for cyclosporin A, 101.6 - 103.0% for sirolimus, 100.0 - 101.2% for tacrolimus, and 99.5 - 102.4% for everolimus. The imprecision of the new measurement procedure, expressed as the coefficient of variation (CV), was 1.17 - 2.60% for cyclosporin A in the concentration range between 8.1 and 979 microg/L, 0.92 - 1.72% for sirolimus in the concentration range between 2.1 and 33.2 microg/L, 0.44 - 1.06% for tacrolimus in the concentration range between 2.0 and 30.8 microg/L and 0.82 - 4.34% for everolimus in the concentration range between 2.1 and 31.4 microg/L.

CONCLUSIONS

An isotope dilution LC-MS/MS procedure for determination of four immunosuppressive drugs was developed to provide a basis for further development toward a reference measurement procedure.

摘要

背景

环孢素A、西罗莫司、他克莫司和依维莫司是用于器官移植后治疗的免疫抑制药物。有多种分析方法可用于监测血液中的药物水平，例如免疫分析法和高效液相色谱-质谱联用（MS）法。从外部质量评估计划中可以明显看出，分析结果显示出高度分散性，需要进一步标准化。

方法

使用pH 9.5的醋酸铵缓冲液和叔丁基甲醚/乙酸乙酯（1:1 v/v）从全血样本中进行液/液萃取药物。通过高效液相色谱法，使用苯基己基反相柱，采用由水、甲醇和含有0.1% v/v甲酸和0.1 mmol/L Cs+的乙腈组成的三元梯度洗脱曲线，实现免疫抑制药物的分离。使用[2H12]-环孢素A、[13C, 2H3]-雷帕霉素、[13C, 2H2]-他克莫司和[13C2, 2H4]-42-O-(2-羟乙基)雷帕霉素作为内标，通过同位素稀释质谱法对免疫抑制药物进行定量分析。

结果

通过分析加标血样来确定新方法的回收率。在加标乙二胺四乙酸（EDTA）全血样本中，环孢素A的回收率为100.8 - 102.5%，西罗莫司为101.6 - 103.0%，他克莫司为100.0 - 101.2%，依维莫司为99.5 - 102.4%。新测量方法的不精密度以变异系数（CV）表示，在8.1至979 μg/L浓度范围内，环孢素A的变异系数为1.17 - 2.60%；在2.1至33.2 μg/L浓度范围内，西罗莫司为0.92 - 1.72%；在2.0至30.8 μg/L浓度范围内，他克莫司为0.44 - 1.06%；在2.1至31.4 μg/L浓度范围内，依维莫司为0.82 - 4.34%。