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首页> 外文会议>American Society for Mass Spectrometry Conference on Mass Spectrometry and Allied Topics >Development of a Sensitive, High-throughput Solid Phase Extraction-Immunoaffinity LC-MS/MS Assay for the Quantification of a Biomarker Peptide, Alpha Calcitonin Gene Related Peptide, in Rat and Human Plasma
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Development of a Sensitive, High-throughput Solid Phase Extraction-Immunoaffinity LC-MS/MS Assay for the Quantification of a Biomarker Peptide, Alpha Calcitonin Gene Related Peptide, in Rat and Human Plasma

机译：敏感，高通量固相提取 - 免疫亲和性LC-MS / MS测定用于定量生物标志物肽，大鼠和人血浆中的生物标志物肽，α钙氨基素基因相关肽

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Purpose: To develop and validate a sensitive, high-throughput solid phase extraction-Immunoaffinity (IA) LC-MS/MS assay for the quantification of a biomarker peptide, alpha calcitonin gene related peptide (alpha-CGRP), in rat and human K_(2)EDTA plasma with a lower limit of quantitation of 10.0 pg/mL. Methods: Plasma samples (500 (mu)L) were first extracted using a 96-well HLB solid phase extraction (SPE) cartridge. 8M guanidine hydrochloride and phosphoric acid solution (10percent, v/v) were added to reduce protein binding, stabilize and increase alpha-CGRP extraction recovery. alpha-CGRP was further isolated from SPE extracts through a 96-well plate based immunoaffinity (IA) capture with acid released step. The LC system was composed of an ACQUITY UPLC BEH Column (C8; 100 X 3.0 mm, 1.7 (mu)m) and the mobile phase was gradient elution with 0.1percent formic acid. The MRM transitions for the LC-MS/MS assay were set at m/z 762.3->911.3 for rat alpha-CGRP and m/z 758.9->919.3 for human alpha-CGRP. Results: The LLOQ was 10.0 pg/mL for human alpha-CGRP and rat alpha-CGRP in plasma. Recovery for SPE was greater than 70percent and the total extraction recovery after SPE and IA treatment was above 40.3percent for both human alpha-CGRP and rat alpha-CGRP from plasma.
机译：目的：为了使敏感，高通量固相提取 - 免疫亲和性（IA）LC-MS / MS测定为量化生物标志物肽，α钙素基因相关肽（α-CGRP），大鼠和人K_ （2）EDTA等离子体，定量下限为10.0 pg / ml。方法：首先使用96孔HLB固相萃取（SPE）盒提取等离子体样品（500（mu）L）。加入8M胍盐酸盐和磷酸溶液（10％，v / v）以降低蛋白质结合，稳定和增加α-CGRP提取恢复。通过酸释放的步骤进一步通过基于96孔板的免疫亲和捕获的96孔板的免疫亲和捕获，从SPE提取物中分离α-CGRP。 LC系统由Acquity UPLC BEH柱组成（C8; 100×3.0mm，1.7（mu）m），并且流动相是用0.1甲酸的梯度洗脱。 LC-MS / MS测定的MRM转变为M / Z 762.3-> 911.3，用于大鼠α-CGRP和M / Z 758.9-> 919.3用于人α-CGRP。结果：LLOQ为10.0 pg / ml，用于血浆中的人α-CGRP和大鼠α-CGRP。 SPE的回收率大于70％，SPE和IA处理后的总提取恢复高于40.3％，用于从等离子体的人α-CGRP和大鼠α-CGRP。

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