LC-MS/MS法测定Beagle犬血浆中泼尼松的浓度

目的：建立一种简便、灵敏的液相色谱串联质谱（LC-MS/MS）方法，测定Beagle犬血浆中泼尼松的浓度，并运用该方法研究泼尼松控释片在健康Beagle犬体内的药代动力学行为。方法：样品预处理采用沉淀蛋白法，内标物为洛索洛芬；采用Venusil ASB C₁₈（2.1 mm×50 mm，3 μm）色谱柱，以甲醇-5 mmol·L^-1醋酸铵水溶液为流动相进行梯度洗脱，流速为0.2 mL·min^-1。离子源为ESI源，采用负离子检测方式，以多反应离子监测（MRM）扫描方式进行检测，检测离子为m/z 357.1→m/z 327.1（泼尼松，待测物），m/z 245.0→m/z 83.0（洛索洛芬，内标物）。6只Beagle犬餐后0.5 h口服给予泼尼松控释片1片（5 mg），于不同时间点分别采集静脉血，测定口服给予泼尼松控释片后Beagle犬血浆中泼尼松的浓度。结果：犬血浆中泼尼松质量浓度在1.00~50.0 μg·L^-1范围内线性关系良好，定量下限为1.00 μg·L^-1，日内和日间精密度的RSD≤8.9%，平均提取回收率在76.4%~87.5%之间，内标的提取回收率为90.6%，基质效应在87.1%~95.9%之间，内标基质效应为105.5%；结论：Beagle犬口服泼尼松控释片5 mg后，主要药动学参数t_1/2为（1.60±0.52）h，k_e为（0.47±0.15）h^-1，C_max为（22.0±2.80）μg·L^-1，T_max为（5.8±0.3）h，AUC_{0-16 h}为（93.5±12.7）μg·h·L^-1。本文建立的方法可用于泼尼松在Beagle犬体内药代动力学行为的研究。

Objective: To develop a simple and sensitive liquid chromatography-tandem mass spectrometry(LC-MS/MS)method for the determination of prednisone concentration in Beagle dog plasma,and to study the pharmacokinetic characteristic of prednisone controlled-release tablets in healthy Beagle dogs by LC-MS/MS. Methods: Protein precipitation was used for the sample pretreatment with loxoprofen as internal standard(IS). The chromatographic separation was performed on a Venusil ASB C₁₈ analytical column(2.1 mm×50 mm,3 μm). Methanol -5 mmol·L^-1 ammonium acetate solution was used as the mobile phase with a gradient elution and the mobile phase was delivered at a flow rate of 0.2 mL·min^-1. Electrospray ionization source(ESI)was operated in negative ion mode. The quantification was performed using multiple reaction monitoring(MRM)mode with the transitions of m/z 357.1→327.1 for prednisone and m/z 245.0→83.0 for loxoprofen(IS). The plasma concentration of prednisone was detected after an oral administration of prednisone controlled-release tablets(5 mg)to 6 Beagle dogs 0.5 h after dinner. Results: The linear calibration curve of prednisone was obtained in the concentration range of 1.00-50.0 μg·L^-1. The LLOQ was confimed to be 1.00 μg·L^-1. The intra-and inter-day precision were both less than 8.9% in terms of relative standard deviation(RSD). The average recoveries were in the range of 76.4%-87.5% for prednisone,and 90.6% for loxoprofen(IS),respectively. The matrix effects were in the range of 87.1%-95.9% for prednisone,and 105.5% for loxoprofen(IS),respectively. Conclusion: The main pharmacokinetic parameters of prednisone in Beagle dogs after oral administration of 5 mg prednisone tablets were as follows:t_1/2=(1.60±0.52)h,k_e=(0.47±0.15)h^-1,C_max=(22.0±2.80)μg·L^-1,T_max=(5.8±0.3)h,AUC_{0-16 h}=(93.5±12.7)μg·h·L^-1. This established LC-MS/MS method can be applied to a pharmacokinetic study of prednisone in Beagle dogs.

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