LC-MS/MS法测定干血斑样品中阿戈美拉汀

Determination of agomelatine in dried blood spots by LC-MS/MS method

目的：建立快速、灵敏的液相色谱-串联质谱（LC-MS/MS）法,测定大鼠干血斑（DBS）样品中的阿戈美拉汀,并应用于阿戈美拉汀大鼠药动学研究。方法：给药后的大鼠新鲜全血40μL点于Whatman 903滤纸上,室温下干燥2 h,放入含干燥剂的密封袋保存。取直径为6 mm的干血斑3片,加入甲醇500μL,超声15 min后,进行液-液萃取,以甲醇-甲酸铵-甲酸为流动相,流速0.20 m L·min-1,采用Grom-Sil 120 ODS-5 ST柱分离,通过电喷雾三重四极杆串联质谱,以多反应监测（MRM）方式进行检测,用于定量的离子反应为m/z 244.1→m/z 185.1（阿戈美拉汀）和m/z 249.2→m/z 187.1（d5-阿戈美拉汀）。结果：测定阿戈美拉汀DBS的线性范围为0.025 0-25.0 ng·m L-1;定量下限为0.025 0 ng·m L-1;精密度和准确度均符合要求。结论：DBS采样法全血用量少,样品储存、运输便利,可用于阿戈美拉汀大鼠药动学研究。

Objective： To develop and validate a liquid chromatograph / tandem mass spectrometric（ LC- MS / MS）method for the determination of agomelatine in samples of dried blood spots（ DBS） and to apply this method to the pharmacokinetic study in rats. Methods： An aliquot of 40 μL of whole blood sample from rats after administration was spotted onto the Whatman 903 sampling filter paper and allowed to dry at ambient temperature for 2 h. The DBS samples were stored in a sealed plastic bag containing desiccant. Three 6- mm diameter disks（ equivalent to c. a.30 μL blood） were collected from the filter paper,and 500 μL of methanol was added followed by sonication for15 min. The samples were chromatographed on a Grom- Sil 120 ODS- 5 ST column. The mobile phase consisted of methanol- ammonium formate- formic acid,at a flow rate of 0. 20 m L ·min- 1. A tandem mass spectrometer equipped with ESI source operated in positive ionization mode was used as the detector. Multiple reaction monitoring（ MRM） with the precursor/product ion combinations of m/z 244. 1→m/z 185. 1 amu and m/z 249. 2→m/z 187. 1amu were used to quantify agomelatine and d5- agomelatine,respectively. Results： The linear range of agomelatine in DBS was 0. 025 0- 25. 0 ng·m L- 1and the lower limit of quantification was 0. 025 0 ng·m L- 1. The precision and accuracy results for the calibration standards and validation samples were within acceptable criteria. Conclusion： The DBS sampling offers the advantages of less sample volume and convenience of sample storage and shipment. The method can be successfully applied to the pharmacokinetic study of agomelatine in rats.