溴吡斯的明血药浓度的HPLC-MS/MS测定及其在人体的药动学研究

Pharmacokinetics of Pyridostigmine Bromide Tablets in Healthy Volunteers Using HPLC-MS/MS Method

目的 建立HPLC-MS/MS测定人血浆中溴吡斯的明的药物浓度，并研究溴吡斯的明片在健康人体内的药动学。方法 采用HPLC-MS/MS检测法，用乙腈沉淀蛋白法处理血浆样本。采用Shimadzu Shim-Pack VP-ODS色谱柱（150 mm×2.0 mm，5 μm）；流动相为甲醇-水（含5 mmol·L-1醋酸铵和0.1%甲酸），梯度洗脱；流速：0.2 mL·min-1；采用三重四级杆质谱仪，电喷雾离子源，正离子模式，选择性离子检测，溴吡斯的明和内标新斯的明的检测离子分别为m/z 181.1→72.2，m/z 223.1→208.1。20名健康受试者口服溴吡斯的明片60 mg后，在不同时间点采集血浆样品，采用建立的HPLC-MS/MS条件测定溴吡斯的明血药浓度，计算药动学参数。结果 溴吡斯的明血药浓度在0.5~100 ng·min-1内样品峰面积比与浓度线性关系良好（r=0.996 9），绝对回收率，日内、日间精密度，冷冻、冻融、室温稳定性均符合生物样品分析要求。20名健康受试者口服溴吡斯的明片60 mg 后的主要药动学参数如下：Cmax为（43.26±10.33）ng·mL-1；Tmax为（1.77±0.48）h；t1/2为（5.11±0.96）h；AUC0-t为（204.8±64.93）ng·h·mL-1；AUC0-∞为（212.42±68.31）ng·h·mL-1。结论 该方法简单、准确、灵敏，适用于溴吡斯的明血药浓度测定和药动学研究。

OBJECTIVE To establish an HPLC-MS/MS method for determination of the concentration of pyridostigmine bromide in human plasma and to study its application on pharmacokinetic. METHODS A liquid chromatography-tandem mass spectrometry method was developed and validated for the determination and pharmacokinetic study of pyridostigmine bromide in human plasma using neostigmine methylsulfate as internal standard （IS）. The chromatographic separation was performed on a SHIMADZU VP-ODS（150 mm×2.0 mm, 5 μm） analytical column using a mobile phase consisting of methanol and water containing 5 mmol·L-1 ammonium acetate and 0.1% formic acid with gradient elution at a ？ow rate of 0.2 mL·min-1. The detection was carried out on a triple quadrupole tandem mass spectrometer by selective reaction monitoring （SRM） via electrospray ionization interface with positive mode, monitoring the transition of the molecular ions m/z 181.1→72.2 for PB and 233.1→208.1 for IS. RESULTS Validation of the method demonstrated that the linear calibration curves covered the range of 0.5-100 ng·mL-1, and the correlation coefficient was 0.996 9. The method was proved to be sensitive, specific, precise, accurate, and robust, revealing that it was appropriate for determination of pyridostigmine bromide in human plasma and successfully applied to the pharmacokinetics study after an oral administration of 60 mg pyridostigmine bromide tablets to twenty Chinese healthy volunteers. The mean of Cmax and Tmax were （43.26±10.33）ng·mL-1 and （1.77±0.48）h, respectively. Plasma concentration declined with a t1/2 of （5.11±0.96）h. AUC0？24 and AUC0？∞ values obtained were （204.8±64.93）ng·h·mL-1 and （212.42± 68.31）ng·h·mL-1, respectively. CONCLUSION This paper describes a sensitive, specific and robust HPLC-MS/MS method for the determination of pyridostigmine bromide in human plasma. The method offeres a simple, economic, rapid and efficient extraction procedure, which is successfully applied to pharmacokinetic studies. The pharmacokinetic parameters in this paper can provide a theoretical basis for clinical medication.