Pigliasco Federica, Cafaro Alessia, Simeoli Raffaele, Barco Sebastiano, Magnasco Alberto, Faraci Maura, Tripodi Gino, Goffredo Bianca Maria, Cangemi Giuliana
Chromatography and Mass Spectrometry Section, Central Laboratory of Analysis, IRCCS Istituto Giannina Gaslini, 16147 Genoa, Italy.
Department of Pediatric Specialties, Division of Metabolic Biochemistry, Children's Hospital Bambino Gesù, IRCCS, 00165 Rome, Italy.
Biomedicines. 2021 Oct 2;9(10):1379. doi: 10.3390/biomedicines9101379.
The role of therapeutic drug monitoring (TDM) of valaciclovir (VA)/aciclovir (A) and valganciclovir/ganciclovir (VG/G) in critically ill patients is still a matter of debate. More data on the dose-concentration relationship might therefore be useful, especially in pediatrics where clinical practice is not adequately supported by robust PK studies. We developed and validated a new liquid chromatography-tandem mass spectrometry (LC-MS/MS) micro-method to simultaneously quantify A and G from plasma and dried plasma spots (DPS). The method was based on rapid organic extraction from DPS and separation on a reversed-phase C-18 UHPLC column after addition of deuterated internal standards. Accurate analyte quantification using SRM detection was then obtained using a Thermo Fisher Quantiva triple-quadrupole MS coupled to an Ultimate 3000 UHPLC. It was validated following international (EMA) guidelines for bioanalytical method validation and was tested on samples from pediatric patients treated with A, VG, or G for cytomegalovirus infection following solid organ or hematopoietic stem cell transplantation. Concentrations obtained from plasma and DPS were compared using Passing-Bablok and Bland-Altman statistical tests. The assay was linear over wide concentration ranges (0.01-20 mg/L) in both plasma and DPS for A and G, suitable for the expected therapeutic ranges for both Cmin and Cmax, accurate, and reproducible in the absence of matrix effects. The results obtained from plasma and DPS were comparable. Using an LC-MS/MS method allowed us to obtain a very specific, sensitive, and rapid quantification of these antiviral drugs starting from very low volumes (50 μL) of plasma samples and DPS. The stability of analytes for at least 30 days allows for cost-effective shipment and storage at room temperature. Our method is suitable for TDM and could be helpful for improving knowledge on PK/PD targets of antivirals in critically ill pediatric patients.
伐昔洛韦（VA）/阿昔洛韦（A）以及缬更昔洛韦/更昔洛韦（VG/G）的治疗药物监测（TDM）在重症患者中的作用仍存在争议。因此，获取更多关于剂量 - 浓度关系的数据可能会有所帮助，尤其是在儿科领域，因为可靠的药代动力学（PK）研究对临床实践的支持尚不充分。我们开发并验证了一种新的液相色谱 - 串联质谱（LC - MS/MS）微量方法，用于同时定量血浆和干血浆斑（DPS）中的A和G。该方法基于从DPS中进行快速有机萃取，并在加入氘代内标后在反相C - 18超高效液相色谱（UHPLC）柱上进行分离。然后使用与Ultimate 3000 UHPLC联用的赛默飞世尔Quantiva三重四极杆质谱仪，通过选择反应监测（SRM）检测实现准确的分析物定量。该方法按照国际（EMA）生物分析方法验证指南进行了验证，并在接受A、VG或G治疗巨细胞病毒感染的儿科患者样本上进行了测试，这些患者均接受了实体器官或造血干细胞移植。使用Passing - Bablok和Bland - Altman统计检验对血浆和DPS获得的浓度进行了比较。该测定法在血浆和DPS中，A和G的浓度范围均很宽（0.01 - 20 mg/L）呈线性，适用于Cmin和Cmax的预期治疗范围，准确且在无基质效应时可重复。从血浆和DPS获得的结果具有可比性。使用LC - MS/MS方法使我们能够从非常少量（50μL）的血浆样本和DPS中，非常特异性、灵敏且快速地定量这些抗病毒药物。分析物至少30天的稳定性允许在室温下进行经济高效的运输和储存。我们的方法适用于TDM，有助于提高对重症儿科患者中抗病毒药物PK/PD靶点的认识。
