National Institute of Occupational Safety and Prevention (ISPESL), Research Center at the University of Parma, Italy.
J Chromatogr B Analyt Technol Biomed Life Sci. 2010 Oct 1;878(27):2616-22. doi: 10.1016/j.jchromb.2010.02.010. Epub 2010 Feb 16.
We present the application of a novel isotope dilution method, named Alternate Isotope-Coded Derivatization Assay (AIDA), to the quantitative analysis of hydrazone derivatives of malondialdehyde (MDA) and 4-hydroxynonenal (4-HNE) in exhaled breath condensate (EBC) samples using liquid chromatography-tandem mass spectrometry. AIDA is based on the alternate derivatization of the analyte(s) with reagents that are available in two pure isotopic forms, respectively "light" and "heavy", by using light-derivatized standards for the quantification of the heavy-derivatized analytes, and vice versa. To this purpose, 2,4-dinitro-3,5,6-trideuterophenylhydrazine (d(3)-DNPH) has been synthesized and used as "heavy" reagent in combination with commercial "light" DNPH. Using the AIDA method, any unknown concentration of the analyte in the matrix can be calculated without the need of a calibration curve. An external calibration method has been also investigated for comparative purpose. The stability of DNPH and d(3)-DNPH derivatives was verified by excluding any exchange of hydrazones with each other. In the range of concentrations of biological interest, e.g., 2-40 nM for MDA and 0.5-10 nM for 4-HNE, the derivatization reactions of MDA and 4-HNE with DNPH and d(3)-DNPH showed overlapping kinetics and comparable yields. The MS response of both DNPH and d(3)-DNPH hydrazones was similar. The precision of AIDA, calculated as %RSD, was within 3.2-8% for MDA and 4.5-11% for 4-HNE. Accuracy was tested by analyzing a spiked EBC pool sample and acceptable results (accuracy within 98-108% for MDA and 93-114% for 4-HNE) were obtained by AIDA after subtraction of the blank, which was not negligible. The results of quantitative analysis of MDA and 4-HNE in EBC samples obtained by AIDA assay with four analyses per sample were in good agreement with those obtained by external calibration method on the same samples.
我们介绍了一种新型的同位素稀释方法，称为交替同位素编码衍生化分析（AIDA），用于通过液相色谱-串联质谱法对呼气冷凝物（EBC）样品中丙二醛（MDA）和 4-羟基壬烯醛（4-HNE）的腙衍生物进行定量分析。AIDA 基于用两种纯同位素形式（分别为“轻”和“重”）的试剂对分析物进行交替衍生化，通过使用轻衍生化标准品对重衍生化分析物进行定量，反之亦然。为此，合成了 2,4-二硝基-3,5,6-三氘苯腙（d(3)-DNPH），并将其与商业“轻”DNPH 一起用作“重”试剂。使用 AIDA 方法，无需校准曲线即可计算基质中任何未知浓度的分析物。还研究了外部校准方法用于比较目的。通过排除腙之间的任何交换，验证了 DNPH 和 d(3)-DNPH 衍生物的稳定性。在生物学感兴趣的浓度范围内，例如 MDA 的 2-40 nM 和 4-HNE 的 0.5-10 nM，MDA 和 4-HNE 与 DNPH 和 d(3)-DNPH 的衍生化反应表现出重叠的动力学和可比的产率。DNPH 和 d(3)-DNPH 腙的 MS 响应相似。AIDA 的精密度，以 %RSD 表示，在 MDA 为 3.2-8%，4-HNE 为 4.5-11%。通过分析加标 EBC 池样品测试准确性，AIDA 在扣除空白后（空白不可忽略）获得了可接受的结果（MDA 的准确性在 98-108%之间，4-HNE 的准确性在 93-114%之间）。通过 AIDA 测定，每个样品进行四次分析，EBC 样品中 MDA 和 4-HNE 的定量分析结果与相同样品的外部校准方法得到的结果非常吻合。
