Department of Drug Analysis, Faculty of Pharmacy, University of Belgrade, Vojvode Stepe 450, 11000 Belgrade, Serbia.
AstraZeneca UK Limited Representative Office, Milutina Milankovića 1i, 11000 Belgrade, Serbia.
Molecules. 2022 Jul 31;27(15):4899. doi: 10.3390/molecules27154899.
In this research, a UHPLC-MS/MS method was developed and validated for the determination of zonisamide in dried plasma spots (DPS) and dried blood spots (DBS). Detection of zonisamide and internal standard, 1-(2,3-dichlorphenyl)piperazine, was carried out in ESI+ mode by monitoring two MRM transitions per analyte. Total run time, less than 2.5 min, was achieved using Acquity UPLC BEH Amide (2.1 × 100 mm, 1.7 µm particle size) column with mobile phase comprising acetonitrile-water (85:15%, /) with 0.075% formic acid. The flow rate was 0.225 mL/min, the column temperature was 30 °C and the injection volume was 3 µL. Desolvation temperature, desolvation gas flow rate, ion source temperature and cone gas flow rate were set by the IntelliStart software tool in combination with tuning. All of the Guthrie cards were scanned, and DPS/DBS areas were determined by the image processing tool. The influence of hematocrit values (20-60%) on accuracy and precision was evaluated to determine the range within which method for DBSs is free from Hct or dependency is within acceptable limits. The validated method was applied to the determination of zonisamide levels in DPS and DBS samples obtained from patients confirming its suitability for clinical application. Finally, the distribution of zonisamide into the red blood cells was estimated by correlating its DPS and DBS levels.
本研究建立并验证了一种 UHPLC-MS/MS 法，用于测定干血浆斑（DPS）和干血斑（DBS）中的佐米曲坦。采用 ESI+ 模式，通过监测每个分析物的两个 MRM 转换，对佐米曲坦和内标 1-（2,3-二氯苯基）哌嗪进行检测。使用 Acquity UPLC BEH Amide（2.1×100mm，1.7µm 粒径）柱，以乙腈-水（85:15%，/）为流动相，其中含有 0.075%的甲酸，总运行时间不到 2.5 分钟。流速为 0.225mL/min，柱温为 30°C，进样量为 3µL。通过 IntelliStart 软件工具与调谐相结合，设定了蒸发温度、蒸发气体流速、离子源温度和锥孔气体流速。所有的 Guthrie 卡都进行了扫描，并通过图像处理工具确定了 DPS/DBS 区域。评估了红细胞比容值（20-60%）对准确度和精密度的影响，以确定 DBS 方法不受 Hct 影响或依赖性在可接受范围内的范围。该验证方法应用于从患者中获得的 DPS 和 DBS 样本中佐米曲坦水平的测定，证实了其适合临床应用。最后，通过比较其 DPS 和 DBS 水平，估计了佐米曲坦在红细胞中的分布。
