Validated liquid chromatography–tandem mass spectrometry method for quantification of ticagrelor and its active metabolite in human plasma

A rapid, simple and sensitive LC–MS/MS method was established and validated for simultaneous quantification of ticagrelor and its active metabolite AR‐C124910XX in human plasma. After plasma samples were deproteinized with acetonitrile, the post‐treatment samples were chromatographed on a Dikma C₁₈ column interfaced with a triple quadrupole tandem mass spectrometer. Electrospray negative ionization mode and multiple reaction monitoring were adopted to assay ticagrelor and AR‐C124910XX. Acetonitrile and 5 mΜ ammonium acetate was used as the mobile phase with a gradient elution at a flow rate of 0.5 mL/min. The method was linear in the range of 0.781–800 ng/mL for both ticagrelor and AR‐C124910XX with a correlation coefficient ≥0.994. The intra‐ and inter‐day precisions were within 12.61% in terms of relative standard deviation and the accuracy was within ±7.88% in terms of relative error. The LC–MS/MS method was fully validated for its sensitivity, selectivity, stability, matrix effect and recovery. This convenient and specific LC–MS/MS method was successfully applied to the pharmacokinetic study of ticagrelor and AR‐C124910XX in healthy volunteers after an oral dose of 90 mg ticagrelor.     

定量核磁共振氢谱测定新药替格瑞洛

本文建立了基于核磁共振氢谱（¹H NMR）测定新药替格瑞洛绝对含量的方法.采用Bruker Avance 300型NMR谱仪,以磺胺多辛为内标;以替格瑞洛中质子信号δ_H 7.14（2H,m）和δ_H 7.04（1H,s）,磺胺多辛质子信号δ_H 8.04（1H,s）、δ_H 7.73（2H,d）和δ_H 6.54（2H,d）作为定量峰;以氘代甲醇（CD₃OD）为溶剂进行测定.测定条件为：探头温度为308 K,谱宽为3 511.5 Hz,中心频率为1 470.6 Hz,脉冲翻转角为θ=30°,延迟时间为10 s,采样次数为16,线宽因子为0.3 Hz.在此实验条件下,替格瑞洛样品与内标磺胺多辛的定量峰分离良好,实验结果精密度较高、重复性较好、线性范围较宽,其线性拟合方程为：Y=1.053X-0.081（r=0.996,n=5）.最终测得样品中替格瑞洛含量为99.4%,相对标准偏差（RSD）为0.20%.该方法简便、准确、快速,适用于替格瑞洛样品的绝对含量测定.     

Development and validation of LC–QTOF–MS/MS method for the identification and determination of low levels of a genotoxic impurity, 4,6-dichloro-5-nitro-2-(propylthio)pyrimidine in ticagrelor API

The foremost objective of the present study was to develop and validate a new LC–QTOF–MS/MS method for the identification and quantitative determination of 4,6-dichloro-5-nitro-2-(propylthio)pyrimidine (DPP) genotoxic impurity through the derivatization process in ticagrelor active pharmaceutical ingredient (API). Owing to the low response of DPP at the specification level, DPP was converted to 4,6-dibenzylamino-5-nitro-2-(propylthio)pyrimidine (DPP derivative) by addition of benzyl amine, then analyzed using mass spectrometry with a time-of-flight analyzer, and good separation was accomplished under the experimental conditions described. The effective separation of DPP derivative was achieved using an Acquity UPLC BEH C₁₈ reverse-phase column (100 × 4.6 mm × 1.7 μm) with an isocratic program with mobile phase A as 0.1% formic acid in milli Q water and mobile phase B as acetonitrile in the ratio of 20:80 v/v. The flow rate was maintained as 0.4 ml/min, the injection volume was 2 μl, the autosampler temperature was 5°C, the column oven temperature was ambient and the run time was 6.0 min. The diluent used was 0.2% benzyl amine in water and acetonitrile in the ratio of 30:70 v/v. The retention time of the DPP derivative was 2.87 min. The limit of detection and limit of quantification were 0.03 and 0.08 ppm, respectively. The DPP derivative was linear from 1.68 to 12.78 ppm with R² of 0.9958. Thus, the developed method is valid for the identification and quantitative determination of DPP derivative in ticagrelor API.     

替格瑞洛的合成工艺改进

以2-丙硫基-4,6-二氯-5-氨基嘧啶(3)、2-[((3aR,4S,6R,6a S)-6-氨基-2,2-二甲基四氢-3a H-环戊基[d][1,3]二氧-4-基)氧]-1-乙醇L-酒石酸盐(4)为起始原料,经亲核取代、环合、与(1R,2S)-2-(3,4-二氟苯基)环丙胺D-扁桃酸盐(2)发生亲核取代、稀盐酸脱保护制得抗血小板药替格瑞洛,所得目标经1H NMR及MS确证,总收率为65%。     

Analytical methodologies for the determination of ticagrelor

Ticagrelor is an orally administered platelet aggregation inhibitor with a cyclopentyl‐triazolopyrimidine structure; it is a selective reversible P2Y12 receptor antagonist, which prevents P2Y12‐mediated and ADP‐mediated platelet activation and aggregation. It is used to reduce the rate of cardiovascular death, myocardial infarction and stroke in patients with acute coronary syndrome or history of myocardial infarction. Several analytical methods have been published for the determination of ticagrelor in pharmaceuticals and biological materials by spectrophotometry, high‐performance liquid chromatography with ultraviolet detection and liquid chromatography coupled with tandem mass spectrometry. The purpose of the current review is to provide a systematic survey of the analytical techniques used for the determination of ticagrelor since its introduction in therapy until today.