HPLC-MS/MS法在骨髓移植患者他克莫司血药浓度测定中的应用

Application of HPLC-MS/MS Method in the Determination of Whole Blood Concentration of Tacrolimus in Bone Marrow Transplant Patient

目的建立测定骨髓移植患者全血他克莫司(FK506)血药浓度的高效液相色谱-质谱联用(HPLC-MS/MS)法,探索HPLC-MS/MS法与羧基金属免疫分析(CMIA)法和化学发光微粒免疫(Elecsys)法在FK506血药浓度检测中的关系,为临床合理选用FK506血药浓度监测方法提供可靠依据。方法采用子囊霉素作内标,全血样品经合内标甲醇沉淀蛋白处理。色谱柱为Ultimate XB-C_(18),柱温65℃,流动相为含0.1 g/dl甲酸和2 mmol/IL乙酸铵的水和含0.1ml/dl甲酸的甲醇,梯度洗脱。质谱检测方式为电喷雾离子阱正离子模式,MRM扫描,监测FK506 m/z 821.5~768.5,子囊霉素m/z809.4~756.4。选取部分骨髓移植患者全血FK506样本,分别用HPLC-MS/MS法,CMIA法和Elecsys法进行检测,对各种检测方法所检测的FK506浓度值进行比较。结果建立的HPLC-MS/MS法检测FK506血药浓度在0.5~50 ng/ml范围内线性关系良好,Y=0.228X-0.003 08(r=0.999 0)。HPLC-MS/MS法与CMIA法和Elecsys法检测FK506血药浓度结果相关性较好,差异无统计学意义。结论建立了检测FK506血药浓度的HPLC-MS/MS法,该法可用于临床骨髓移植患者全血FK506浓度的监测。

Objective To develop a HPLC-MS/MS method for determination of Tacrolimus（FKS06） in bone marrow trans plant patient,and explore the relationship of HPLC-MS/MS with CMIA and Elecsys in the measurement of FK506 blood drug concentration,and provide reliable basis for clinical rational use of monitoring method in FKS06 blood drug coneentra tion. Methods The separation was performed on a Ultimate XB-C18 column with a mobile phase of water （containing 2 mmol/L ammonium acetate and 0.1 ml/dl formic acid） and methanol （containing 0.1 ml/dl formic acid）. The way of eluting was gradient. Mass spectrum detection method was ESI positive ion mode and the MRM transitions of FK506 m/z 821.5～ 768.5 and Ascomycin m/z 809.4～756.4. Selected part of the whole blood samples of FK506 in bone marrow transplant pa- tient and respectively were tested by HPLC/MS/MS method, CMIA method and Elecsys method. Then, three methods for the detection of FKS06 density were compared. Results The standard curve of FK506 was linear over the range of 0.5～50 ng/ml,Y=0. 228X-0. 003 08 （r=0. 999 0）. FK506 blood concentrations were not significantly different in HPLC/MS/MS method,CMIA method and Elecsys method. Conclusion The HPLC-MS/MS method in the detection of FK506 was estab- lished and can be used for monitoring of whole blood concentration of FK506 in bone marrow transplant patients.