Biomarker Research, Pharmacokinetics, Dynamics and Metabolism, Pfizer Global Research and Development, Groton, CT 06340, USA.
Cardiovascular, Metabolic, and Endocrine Diseases Research Unit, Pfizer Global Research and Development, Cambridge, MA 02139, USA.
J Chromatogr A. 2014 Jan 17;1325:129-36. doi: 10.1016/j.chroma.2013.12.017. Epub 2013 Dec 16.
We present here a novel and highly sensitive ion-pair hydrophilic interaction chromatography-tandem mass spectrometry (IP-HILIC-MS/MS) method for quantitation of highly polar acid metabolites like adenine nucleotides. A mobile phase based on diethylamine (DEA) and hexafluoro-2-isopropanol (HFIP) and an aminopropyl (NH2) column were applied for a novel chromatographic separation for the determination of AMP, ADP and ATP in biological matrices. This novel IP-HILIC mechanism could be hypothesized by the ion-pairing reagent (DEA) in the mobile phase forming neutral and hydrophilic complexes with the analytes of polar organic acids. The IP-HILIC-MS/MS assay for adenine nucleotides was successfully validated with satisfactory linearity, sensitivity, accuracy, reproducibility and matrix effects. The lower limit of quantitation (LLOQ) at 2.00ng/mL obtained for ATP showed a least 10-fold higher sensitivity than previous LC-MS/MS assays except nano-LC-MS/MS assay. In summary, this novel IP-HILIC-MS/MS assay provides a sensitive method for nucleotides bioanalysis and shows great potential to determine a number of organic acids in biological matrices.
我们在此提出了一种新颖且高灵敏度的离子对亲水性相互作用色谱-串联质谱（IP-HILIC-MS/MS）方法，用于定量分析高度极性的酸性代谢物，如腺嘌呤核苷酸。该方法采用基于二乙胺（DEA）和六氟-2-异丙醇（HFIP）的流动相和氨基丙基（NH2）柱，用于生物基质中 AMP、ADP 和 ATP 的新型色谱分离测定。这种新型的 IP-HILIC 机制可以通过流动相中的离子对试剂（DEA）与极性有机酸的分析物形成中性和亲水复合物来假设。腺嘌呤核苷酸的 IP-HILIC-MS/MS 测定法经过验证，具有令人满意的线性、灵敏度、准确性、重现性和基质效应。对于 ATP，LLOQ 在 2.00ng/mL 时的灵敏度比以前的 LC-MS/MS 测定法高出至少 10 倍，除了纳升 LC-MS/MS 测定法。总之，这种新型的 IP-HILIC-MS/MS 测定法为核苷酸的生物分析提供了一种灵敏的方法，并且在确定生物基质中的多种有机酸方面显示出很大的潜力。
