同时测定血浆中琥乙红霉素和活性代谢产物的LC-MS/MS同位素内标法的建立及药代动力学研究

目的：运用同位素内标，建立同时测定体内琥乙红霉素及其活性代谢产物红霉素的LC-MS/MS检测方法，并进行比格犬体内的药代动力学研究。方法：以氘代同位素（红霉素-D6）为内标，血浆样品经乙腈蛋白沉淀处理后，采用LC-MS/MS法同时测定琥乙红霉素及红霉素的血药浓度。色谱条件：采用Thermo Hypersil GOLD色谱柱（2.1 mm×150 mm，3 μm），以10 mmol·L^-1甲酸铵水溶液-乙腈（60∶40，含0.5%甲酸）为流动相，流速0.3 mL·min^-1；质谱参数：电喷雾离子源（ESI），选择离子监测（SRM模式），监测离子对为m/z 734.486→m/z 157.906（红霉素）、m/z 862.522→ m/z 285.722（琥乙红霉素）、m/z 740.509→m/z 163.932（红霉素-D6）。结果：本方法检测血浆中琥乙红霉素及红霉素的线性范围分别为4~4 000 ng·mL^-1（r=0.999 8）、2~1 000 ng·mL^-1（r=0.999 9），定量下限分别为4、2 ng·mL^-1，相对误差（RE）分别为-1.17%~4.92%、-2.20%~6.44%，批内及批间RSD分别为3.28%~9.43%、0.91%~6.10%，提取回收率分别为98.3%~107.9%、97.9%~103.4%，内标归一化的基质因子RSD分别为2.18%~6.77%、0.36%~3.68%。比格犬灌胃给予琥乙红霉素混悬剂每只200 mg，体内琥乙红霉素T_1/2为（3.50±2.43）h，C_max为（1 466.24±479.49）ng·mL^-1，AUC_（0-t）为（1 080.18±311.86）μg·L^-1·h^-1；红霉素T_1/2为（6.46±5.49）h，C_max为（233.34±83.78）ng·mL^-1，AUC_（0-t）为（324.09±58.85）μg·L^-1·h^-1。结论：本方法简便、灵敏、高效，特异性强，可用于体内琥乙红霉素和活性代谢产物红霉素的同时检测及其药代动力学研究。

Objective: To establish a LC-MS/MS method with isotope internal standards for the simultaneous determination of erythromycin ethylsuccinate and its active metabolite in plasma,and to study the pharmacokinetics of beagle dogs. Methods: Using deuterium labeled isotope (erythromycin-D6) as internal standard,plasma samples were precipitated by acetonitrile. Erythromycin ethylsuccinate and its active metabolite in plasma were detected simultaneously by LC-MS/MS. The separation was performed on the Thermo Hypersil GOLD column (2.1 mm×150 mm,3 μm) with a mixture of 10 mmol·L^-1 ammonium formate solution-acetonitrile (60:40,containing 0.5% formic acid) as the mobile phase at flow rate of 0.3 mL·min^-1. LC-MS/MS system (SRM mode) and ESI was used. The ions of monitor were m/z 734.486→m/z 157.906 (erythromycin),m/z 862.522→m/z 285.722 (erythromycin ethylsuccinate),m/z 740.509→m/z 163.932 (erythromycin-D6). Results: The linear range of the standard curve of erythromycin ethylsuccinate and erythromycin were 4~4 000 ng·mL^-1 (r=0.999 8),2~1 000 ng·mL^-1 (r=0.999 9),the limit of detection were 4 ng·mL^-1,2 ng·mL^-1 respectively. Relative errors (RE) were -1.17%-4.92%,-2.20%-6.44% respectively. The within-run and between-run RSDs were 3.28%-9.43%,0.91%-6.10% respectively. The recovery rates of erythromycin ethylsuccinate and erythromycin were 98.3%-107.9%,97.9%-103.4% respectively. RSDs of internal standard normalized matrix factors were 2.18%-6.77%,0.36%-3.68% respectively. Each beagle dog was given 200 mg of erythromycin succsuccinate suspension by gavage administration. The main pharmacokinetic parameters of erythromycin ethylsuccinate were estimated to be as follows:T_1/2= (3.50±2.43) h,C_max= (1 466.24±479.49) ng·mL^-1,AUC_(0-t)=(1 080.18±311.86) μg·L^-1·h^-1;the main pharmacokinetic parameters of erythromycin were estimated to be as follows:T_1/2= (6.46±5.49) h,C_max= (233.34±83.78) ng·mL^-1,AUC_(0-t)=(324.09±58.85) μg·L^-1·h^-1. Conclusion: The method is simple,efficient,sensitive and specific and can be applied to the simultaneous detection and pharmacokinetic study of erythromycin ethylsuccinate and erythromycin.

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