Department of Laboratory Medicine, Bambino Gesù Children's Research Hospital, IRCCS, Rome, Italy.
Department of Laboratory Medicine, Bambino Gesù Children's Research Hospital, IRCCS, Rome, Italy.
Clin Chim Acta. 2015 Feb 2;440:108-12. doi: 10.1016/j.cca.2014.11.014. Epub 2014 Nov 15.
Pipecolic acid (PA) is an important biochemical marker for the diagnosis of peroxisomal disorders. PA is also a factor responsible for hepatic encephalopathy and a possible biomarker for pyridoxine-dependent seizures. We developed an easy and rapid PA quantification method, by high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS), requiring no derivatization and applicable to small sample volumes. Plasma (100 μl) is extracted with 500 μl acetonitrile (ACN) containing 2 μmol/l [(2)H5]-phenylalanine as internal standard, vortexed and centrifuged. The supernatant is analyzed by HPLC-MS/MS in positive-ion mode using multiple reaction monitoring scan type. HPLC column is a Luna HILIC (150×3.0mm; 3 μ 200A): Buffer A: ammonium formate 5 mmol/l; Buffer B: ACN/H20 90:10 containing ammonium formate 5 mmol/l. PA retention time is 4.86 min. Recovery was 93.8%, linearity was assessed between 0.05 and 50 μmol/l (R(2)=0.998), lower limit of detection was 0.010 μmol/l and lower limit of quantification was 0.050 μmol/l. Coefficient of variation was 3.2% intra-assay and 3.4% inter-assay, respectively. Clinical validation was obtained by comparing PA plasma values from 5 patients affected by peroxisomal disorders (mean, 23.38 μmol/l; range, 11.20-37.1 μmol/l) to 24 ages related healthy subjects (mean, 1.711 μmol/l; range, 0.517-3.580 μmol/l).
哌可酸（PA）是诊断过氧化物酶体疾病的重要生化标志物。PA 还是肝性脑病的一个因素，也是吡哆醇依赖型癫痫的可能生物标志物。我们开发了一种简单快速的 PA 定量方法，采用高效液相色谱-串联质谱（HPLC-MS/MS），无需衍生化，适用于小体积样本。血浆（100 μl）用 500 μl 含 2 μmol/L [（2）H5]-苯丙氨酸的乙腈（ACN）提取，涡旋并离心。上清液在正离子模式下通过 HPLC-MS/MS 以多重反应监测扫描方式分析。HPLC 柱为 Luna HILIC（150×3.0mm；3 μ 200A）：缓冲液 A：5 mmol/L 甲酸铵；缓冲液 B：90:10 的 ACN/H20 中含 5 mmol/L 甲酸铵。PA 保留时间为 4.86 min。回收率为 93.8%，线性评估范围为 0.05-50 μmol/L（R²=0.998），检测限为 0.010 μmol/L，定量限为 0.050 μmol/L。日内和日间变异系数分别为 3.2%和 3.4%。通过比较 5 例过氧化物酶体疾病患者（平均 23.38 μmol/L；范围 11.20-37.1 μmol/L）和 24 例年龄相关健康对照者（平均 1.711 μmol/L；范围 0.517-3.580 μmol/L）的血浆 PA 值，获得了临床验证。
