Mathew Elizabeth Mary, Moorkoth Sudheer, Lewis Leslie, Rao Pragna
Department of Pharmaceutical Quality Assurance, Manipal College of Pharmaceutical Sciences, Manipal Academy of Higher Education, Manipal 576104, India.
Department of Paediatrics, Kasturba Medical College, Manipal Academy of Higher Education, Manipal 576104, India.
Int J Anal Chem. 2018 Aug 1;2018:2583215. doi: 10.1155/2018/2583215. eCollection 2018.
Pyridoxine dependent epilepsy is a condition where the affected infant or child has prolonged seizures (status epilepticus), which are nonresponsive to anticonvulsant therapy but can be treated with pharmacological doses of pyridoxine. If identified earlier and treated prophylactically with pyridoxine, severe brain damage due to seizures can be prevented. Alpha-amino adipic semialdehyde (AASA), piperidine-6-carboxylic acid (P6C), and pipecolic acid (PA) are known biomarkers of pyridoxine dependent epilepsy. We report the development and validation of a hydrophilic interaction liquid chromatography (HILIC) hyphenated with mass spectroscopy for the quantification of the above analytes from dried blood spot samples. The samples were extracted using methanol and analysed on a iHILIC fusion plus column with formic acid buffer (pH 2.5): acetonitrile (20:80) at a flow rate of 0.5 mL/min within 3 minutes. The method demonstrated a LOD of 10 ng/mL, LOQ of 50 ng/mL, linearity of r ≥ 0.990, and recovery of 92-101.98% for all analytes. The intra- and interday precision CVs were < 8% and 6%, respectively. Extensive stability studies demonstrated that the analytes were stable in stock solution and in matrix when stored at -80°C. We performed method comparison studies of the developed method with the literature reported method using normal samples and matrix matched spiked samples at pathological concentrations to mimic clinical validity. The Bland-Altman analysis for comparison of the analytical suitability of the method for the biomarkers in healthy and spiked samples with the literature reported method revealed a bias which suggested that the method was comparable. The newly developed method involves no derivatisation and has a simple sample preparation and a low run time enabling it to be easily automated with a high sample throughput in a cost-effective manner.
吡哆醇依赖性癫痫是一种病症，患病婴儿或儿童会出现长时间的癫痫发作（癫痫持续状态），这些发作对抗惊厥治疗无反应，但可用药理剂量的吡哆醇进行治疗。如果能早期识别并用吡哆醇进行预防性治疗，可预防因癫痫发作导致的严重脑损伤。α-氨基己二酸半醛（AASA）、哌啶-6-羧酸（P6C）和哌可酸（PA）是已知的吡哆醇依赖性癫痫生物标志物。我们报告了一种亲水相互作用液相色谱（HILIC）与质谱联用方法的开发与验证，用于定量测定干血斑样本中的上述分析物。样本用甲醇提取，在iHILIC fusion plus柱上进行分析，流动相为甲酸缓冲液（pH 2.5）：乙腈（20:80），流速为0.5 mL/min，在3分钟内完成分析。该方法对所有分析物的检测限为10 ng/mL，定量限为50 ng/mL，线性相关系数r≥0.990，回收率为92 - 101.98%。日内和日间精密度变异系数分别<8%和6%。广泛的稳定性研究表明，分析物在储存于-80°C的储备溶液和基质中均稳定。我们使用正常样本和病理浓度的基质匹配加标样本进行方法比较研究，将开发的方法与文献报道的方法进行比较，以模拟临床有效性。通过Bland-Altman分析比较该方法与文献报道方法对健康样本和加标样本中生物标志物的分析适用性，结果显示存在偏差，表明该方法具有可比性。新开发的方法无需衍生化，样品制备简单，运行时间短，能够以具有成本效益的方式轻松实现自动化，且样品通量高。
