Lord Heather L, Rosenfeld Jack, Volovich Vitaly, Kumbhare Dinesh, Parkinson Bill
Department of Pathology and Molecular Medicine, McMaster University, Hamilton, ON, Canada.
J Chromatogr B Analyt Technol Biomed Life Sci. 2009 May 1;877(13):1292-8. doi: 10.1016/j.jchromb.2008.12.035. Epub 2008 Dec 24.
Analytical derivatization (AD) increases the sensitivity of analysis by one to three orders of magnitude, stabilizes labile analytes and converts them into readily extractable products. Using a variant of this technique, we applied solid phase analytical derivatization (SPAD) to fully automate extraction, derivatization and liquid chromatography. The resulting device (AutoSPAD) determined malonyldialdehyde (MDA) from biological fluids. This biomarker of oxidative stress is highly water-soluble (500 g/L at pH 7), chemically labile and lacks any functionality that enables detection at high sensitivity. AutoSPAD utilizes column-switching technology to load DANSYL hydrazine onto the solid phase, pass the biological sample over the resulting reactor bed for derivatization on the surface to form a hydrophobic derivative suitable for increasing sensitivity of any other LC technique including LC-MS/MS. The hydrophobic solid phase retains the derivative during washing steps, following which AutoSPAD transfers the derivatized extract to the analytical column for separation and detection by fluorescence. In plasma, however, MDA exists both in free form and covalently bound to protein. Measuring MDA from plasma, therefore, required identification of appropriate protein precipitation and hydrolysis conditions. Under these conditions, the DANSYL derivative formed at only one aldehydic position but did not cyclize as reported for other reactions between hydrazine reagents and MDA. The calibration curve using approximately 7 microL of plasma was linear (r(2)=0.999) in the physiological range (0.1-3 microg/mL) and the relative standard deviation of replicate determinations at 1 microg/mL was less than 5%.
分析衍生化（AD）可将分析灵敏度提高一至三个数量级，稳定不稳定的分析物，并将其转化为易于提取的产物。我们采用该技术的一种变体，应用固相分析衍生化（SPAD）实现提取、衍生化和液相色谱的完全自动化。所得装置（自动SPAD）可测定生物流体中的丙二醛（MDA）。这种氧化应激生物标志物高度水溶性（pH 7时为500 g/L），化学性质不稳定，且缺乏任何能实现高灵敏度检测的功能。自动SPAD利用柱切换技术将丹磺酰肼加载到固相上，使生物样品通过所得反应床在表面进行衍生化，形成适合提高包括液相色谱-串联质谱（LC-MS/MS）在内的任何其他液相色谱技术灵敏度的疏水衍生物。疏水固相在洗涤步骤中保留衍生物，随后自动SPAD将衍生化提取物转移至分析柱进行荧光分离和检测。然而，在血浆中，MDA既以游离形式存在，也以共价键结合到蛋白质上。因此，从血浆中测量MDA需要确定合适的蛋白质沉淀和水解条件。在这些条件下，丹磺酰衍生物仅在一个醛基位置形成，但未像肼试剂与MDA之间的其他反应那样环化。使用约7微升血浆的校准曲线在生理范围（0.1 - 3微克/毫升）内呈线性（r(2)=0.999），1微克/毫升时重复测定的相对标准偏差小于5%。
