Hawwa Ahmed F, Albawab Abdelqader, Rooney Madeleine, Wedderburn Lucy R, Beresford Michael W, McElnay James C
Clinical and Practice Research Group, School of Pharmacy, Queen's University Belfast, Belfast, United Kingdom ; Aston Pharmacy School, Aston University, Birmingham, United Kingdom.
Clinical and Practice Research Group, School of Pharmacy, Queen's University Belfast, Belfast, United Kingdom.
PLoS One. 2014 Feb 25;9(2):e89908. doi: 10.1371/journal.pone.0089908. eCollection 2014.
Development and validation of a selective and sensitive LCMS method for the determination of methotrexate polyglutamates in dried blood spots (DBS).
DBS samples [spiked or patient samples] were prepared by applying blood to Guthrie cards which was then dried at room temperature. The method utilised 6-mm disks punched from the DBS samples (equivalent to approximately 12 µl of whole blood). The simple treatment procedure was based on protein precipitation using perchloric acid followed by solid phase extraction using MAX cartridges. The extracted sample was chromatographed using a reversed phase system involving an Atlantis T3-C18 column (3 µm, 2.1 × 150 mm) preceded by Atlantis guard column of matching chemistry. Analytes were subjected to LCMS analysis using positive electrospray ionization.
The method was linear over the range 5-400 nmol/L. The limits of detection and quantification were 1.6 and 5 nmol/L for individual polyglutamates and 1.5 and 4.5 nmol/L for total polyglutamates, respectively. The method has been applied successfully to the determination of DBS finger-prick samples from 47 paediatric patients and results confirmed with concentrations measured in matched RBC samples using conventional HPLC-UV technique.
The methodology has a potential for application in a range of clinical studies (e.g. pharmacokinetic evaluations or medication adherence assessment) since it is minimally invasive and easy to perform, potentially allowing parents to take blood samples at home. The feasibility of using DBS sampling can be of major value for future clinical trials or clinical care in paediatric rheumatology.
开发并验证一种用于测定干血斑（DBS）中甲氨蝶呤多聚谷氨酸盐的选择性和灵敏的液相色谱-质谱联用（LCMS）方法。
通过将血液涂覆在Guthrie卡片上制备DBS样本（加标样本或患者样本），然后在室温下干燥。该方法使用从DBS样本上冲压出的6毫米圆盘（相当于约12微升全血）。简单的处理程序基于使用高氯酸进行蛋白质沉淀，随后使用MAX柱进行固相萃取。提取的样品使用反相系统进行色谱分析，该系统包括一根Atlantis T3-C18柱（3微米，2.1×150毫米），前面有一根化学性质匹配的Atlantis保护柱。分析物采用正电喷雾电离进行LCMS分析。
该方法在5-400纳摩尔/升范围内呈线性。单个多聚谷氨酸盐的检测限和定量限分别为1.6和5纳摩尔/升，总多聚谷氨酸盐的检测限和定量限分别为1.5和4.5纳摩尔/升。该方法已成功应用于47名儿科患者的DBS指尖采血样本的测定，结果通过使用传统高效液相色谱-紫外（HPLC-UV）技术在匹配的红细胞样本中测得的浓度得到证实。
该方法具有在一系列临床研究（如药代动力学评估或用药依从性评估）中应用的潜力，因为它微创且易于操作，有可能让家长在家中采集血样。使用DBS采样的可行性对于未来儿科风湿病学的临床试验或临床护理可能具有重要价值。
