LC-MS/MS法测定尿二棕榈酰磷脂酰胆碱含量的方法学建立及其作为肾损伤标志物的研究(英文)

Development and validation of an LC-MS/MS method for quantification of dipalmitoylphosphatidylcholine as a promising biomarker for renal failure in urine

尿磷脂已被证实是非常灵敏的肾损伤标志物。然而,关于磷脂的研究大部分为定性或相对定量研究。因此,我们建立了定量测定尿中二棕榈酰磷脂酰胆碱(DPPC)的方法学来准确、特异地测定尿中DPPC含量。采用HILIC Silica(2.1 mm×50 mm,5μm)色谱柱,流动相为5 m M的甲酸铵–乙腈进行梯度洗脱,流速0.3 m L/min。该方法在2–200 ng/m L范围内线性良好,精密度、准确性、回收率、稳定性、稀释效应、基质效应等均符合FDA关于生物样本的检测要求。该方法应用于尿中DPPC的含量测定,并对DPPC作为肾损伤标志物的前景进行评价。

Urinary phospholipids are shown to be sensitive biomarkers for kidney injury. Many works have been reported on qualitative analysis of phospholipids and relative components in human urine while quantitative analysis is rare. We have therefore developed a reliable and convenient quantitative method to evaluate the accuracy and specificity of dipalmitoylphosphatidylcholine （DPPC） as a biomarker for kidney injury diagnosis. Chromatographic separation was achieved using a HILIC Silica Column （2.1 ram^50 mm, 5 p.m）. Gradient elution was performed with 5 mM ammonium formate （0.1% formic acid） and acetonitrile （0.1% formic acid）, at a flow rate of 0.3 mL/min. This method was validated in a linear range of 2-200 ng/mL DPPC with inter-day precision of less than 6.5% and accuracy within 111.2% in human urine. Recovery rate, stability, dilution effect and matrix effect also met the requirements for drug evaluation and research issued by FDA. As the first HPLC-MS/MS method for quantitative determination of DPPC, it has been successfully applied to determine levels of DPPC in clinical urine samples and evaluated for potential use in the measurement of DPPC as a biomarker for kidney injury.