Yeh Li-Tain, Nguyen Mai, Dadgostari Sohelia, Bu Wei, Lin Chin-Chung
Drug Development Department, Valeant Research & Development, 3300 Hyland Avenue, Costa Mesa, CA 92626, USA.
J Pharm Biomed Anal. 2007 Feb 19;43(3):1057-64. doi: 10.1016/j.jpba.2006.09.001. Epub 2006 Oct 6.
A high performance liquid chromatography-tandem mass spectrometry (LC-MS/MS) method has been developed for the simultaneous determinations of total viramidine (viramidine, viramidine monophosphate, viramidine diphosphate, and viramidine triphosphate) and total ribavirin (ribavirin, ribavirin monophosphate, ribavirin diphosphate, and ribavirin triphosphate) in monkey red blood cells (RBC). The method involves the addition of internal standards and perchloric acid, conversion of viramidine or ribavirin phosphorylated metabolites to viramidine or ribavirin, purification with an aminopropyl (NH(2)) solid phase extraction (SPE) cartridge, and LC-MS/MS analysis. The MS/MS is selected to monitor m/z 245-->113, 250-->113, 244-->112, and 249-->112 for ribavirin, [(13)C]ribavirin, viramidine, and [(13)C]viramidine, respectively, using positive electrospray ionization. The calibration curves are linear over a concentration range of 100-10,000 ng/mL (0.412-41.2 microM) with a lower limit of quantification (LLOQ) of 100 ng/mL for both compounds. Mean inter-assay recoveries for ribavirin are 101%, 98.9%, and 96.0%, with coefficient of variance (%CV) values between 1.95 and 4.50% for 100, 1000, and 10,000 ng/mL quality control (QC) samples, respectively. Mean inter-assay recoveries for viramidine are 96.3%, 101%, and 102%, with coefficient of variation (%CV) values between 3.61 and 7.22%, for 100, 1000, and 10,000 ng/mL QC samples, respectively. Over-curve dilution QC at 400 microg/mL (1639 microM) for both viramidine and ribavirin are used to ensure the dilution accuracy (25 X dilutions) for monkey samples. The method has been used to simultaneously determine the total concentrations of ribavirin and viramidine in monkey RBC following 5, 15, and 36 weeks dosing of viramidine or ribavirin (60 mg/kg). The concentrations of total ribavirin following ribavirin dosing are 1242 microM at week 5, 1257 microM at week 15, and 1146 microM at week 36. The concentrations of total ribavirin following viramidine dosing are 634 microM at week 5, 716 microM at week 15, and 683 microM at week 36. Only small amounts of viramidine are detected in RBC following viramidine dosing, 7.80 microM at week 5, 6.63 microM at week 15, and 10.4 microM at week 36. The results suggest that ribavirin levels in RBC were at steady state at week 5 of ribavirin or viramidine dosing. At steady state, ribavirin levels in RBC are approximately 2x after ribavirin dosing than viramidine dosing. The relatively small percentage of viramidine in RBC suggests that viramidine either poorly penetrated into RBC or was extensively converted to ribavirin following entry into RBC.
已开发出一种高效液相色谱 - 串联质谱（LC - MS/MS）方法，用于同时测定猴红细胞（RBC）中总维拉美定（维拉美定、维拉美定单磷酸酯、维拉美定二磷酸酯和维拉美定三磷酸酯）和总利巴韦林（利巴韦林、利巴韦林单磷酸酯、利巴韦林二磷酸酯和利巴韦林三磷酸酯）。该方法包括添加内标和高氯酸，将维拉美定或利巴韦林的磷酸化代谢物转化为维拉美定或利巴韦林，用氨丙基（NH₂）固相萃取（SPE）柱进行纯化，以及LC - MS/MS分析。使用正电喷雾电离，选择MS/MS分别监测利巴韦林、[¹³C]利巴韦林、维拉美定和[¹³C]维拉美定的m/z 245→113、250→113、244→112和249→112。校准曲线在100 - 10,000 ng/mL（0.412 - 41.2 μM）的浓度范围内呈线性，两种化合物的定量下限（LLOQ）均为100 ng/mL。对于100、1000和10,000 ng/mL的质量控制（QC）样品，利巴韦林的批间平均回收率分别为101%、98.9%和96.0%，变异系数（%CV）值在1.95%至4.50%之间。对于100、1000和10,000 ng/mL的QC样品，维拉美定的批间平均回收率分别为96.3%、101%和102%，变异系数（%CV）值在3.61%至7.22%之间。使用维拉美定和利巴韦林在400 μg/mL（1639 μM）的超曲线稀释QC来确保猴样品的稀释准确性（25倍稀释）。该方法已用于在维拉美定或利巴韦林（60 mg/kg）给药5、15和36周后同时测定猴RBC中利巴韦林和维拉美定的总浓度。利巴韦林给药后，第5周总利巴韦林浓度为1242 μM，第15周为1257 μM，第36周为1146 μM。维拉美定给药后，第5周总利巴韦林浓度为634 μM，第15周为716 μM，第36周为683 μM。维拉美定给药后，在RBC中仅检测到少量维拉美定，第5周为7.80 μM，第15周为6.63 μM，第36周为10.4 μM。结果表明，在利巴韦林或维拉美定给药第5周时，RBC中的利巴韦林水平处于稳态。在稳态时，利巴韦林给药后RBC中的利巴韦林水平比维拉美定给药后高约2倍。RBC中维拉美定的比例相对较小，这表明维拉美定要么很难渗透到RBC中，要么进入RBC后被大量转化为利巴韦林。
