Salgado P, Visnevschi-Necrasov T, Kiene R P, Azevedo I, Rocha A C S, Almeida C M R, Magalhães C
CIMAR/CIIMAR - Centre of Marine and Environmental Research, University of Porto, Rua dos Bragas, n° 289, 4050-123 Porto, Portugal.
CIMAR/CIIMAR - Centre of Marine and Environmental Research, University of Porto, Rua dos Bragas, n° 289, 4050-123 Porto, Portugal.
J Chromatogr B Analyt Technol Biomed Life Sci. 2015 Jun 15;992:103-8. doi: 10.1016/j.jchromb.2015.04.008. Epub 2015 Apr 20.
The organic sulfur compound 3-mercaptopropionic acid (3-MPA) is an important thiol intermediate in organic sulfur metabolism in natural environments. It is generated during degradation of sulfur-containing amino acids (e.g. methionine) and from demethylation of dimethylsulfoniopropionate (DMSP). This pathway is an alternative enzymatic process in the DMSP catabolism that routes sulfur away from the climatically-active dimethyl sulfide (DMS). 3-MPA detection and subsequent quantification in different matrices is difficult due to its extreme reactivity. We therefore developed a sensitive method for determination of 3-MPA based on pre-column derivatization with monobromobimane and analysis by high-performance liquid chromatography (HPLC) with fluorescence detection. This methodology was first tested with 3-MPA standards under low (0.005-0.2μmolL(-1)) and high (1-25μmolL(-1)) concentrations. For the optimization of the reaction, CHES and, alternatively, Tris-HCl buffers were evaluated in the derivatization step, with Tris-HCl showing more effective separation of thiol derivatives and a better 3-MPA peak shape. The detection limit was 4.3nmolL(-1) with a 10μL sample injection, and mean recoveries of 3-MPA ranged from 97 to 105% in estuarine waters with different salinities (0.17 and 35.9ppt). The linearity (r>0.99) and repeatability of detector response, with intra- and inter-day precision (% CV) of 2.68-7.01% and 4.86-12.5%, respectively, confirmed the reliability of the method. Previous 3-MPA analytical methods required immediate analysis due to unstable derivatives, but in this method we achieved high stability of the derivatized samples when stored at 4°C, with only a 3-5% loss after more than one year of storage. This method was successfully applied to measure 3-MPA concentrations and rates of 3-MPA production in a variety of intertidal estuarine sediment slurries. Dissolved 3-MPA concentrations in these sediment slurries varied between 2 and 237μmolL(-1) and, 3-MPA net fluxes ranged in wet sediments between -3.6±1.7 and 30±5μmolL(-1)g(-1)h(-1). Thus, the application of this optimized methodology showed an efficient performance for measuring 3-MPA in environmental samples, with a straightforward sample derivatization and a simple analysis of stable 3-MPA derivatives.
有机硫化合物3-巯基丙酸（3-MPA）是自然环境中有机硫代谢的一种重要硫醇中间体。它在含硫氨基酸（如蛋氨酸）的降解过程中以及二甲基巯基丙酸内盐（DMSP）的去甲基化过程中产生。该途径是DMSP分解代谢中的一种替代酶促过程，可将硫从具有气候活性的二甲基硫醚（DMS）中转移出来。由于其极高的反应活性，在不同基质中检测和随后定量3-MPA很困难。因此，我们开发了一种基于用单溴代联苯胺进行柱前衍生化并通过带荧光检测的高效液相色谱（HPLC）分析来测定3-MPA的灵敏方法。该方法首先在低浓度（0.005 - 0.2μmolL⁻¹）和高浓度（1 - 25μmolL⁻¹）的3-MPA标准品上进行测试。为了优化反应，在衍生化步骤中评估了CHES以及Tris - HCl缓冲液，结果表明Tris - HCl对硫醇衍生物的分离更有效，3-MPA峰形更好。进样量为10μL时检测限为4.3nmolL⁻¹，在不同盐度（0.17和35.9ppt）的河口水中3-MPA的平均回收率在97%至105%之间。检测器响应的线性（r > 0.99）以及日内和日间精密度（%CV）分别为2.68 - 7.01%和4.86 - 12.5%，证实了该方法的可靠性。以前的3-MPA分析方法由于衍生物不稳定需要立即分析，但在本方法中，衍生化后的样品在4°C储存时具有很高的稳定性，储存一年多后损失仅为3 - 5%。该方法已成功应用于测量各种潮间带河口沉积物浆液中3-MPA的浓度和3-MPA的产生速率。这些沉积物浆液中溶解的3-MPA浓度在2至237μmolL⁻¹之间，在湿沉积物中3-MPA的净通量在-3.6±1.7至30±5μmolL⁻¹g⁻¹h⁻¹之间。因此，这种优化方法的应用在测量环境样品中的3-MPA方面表现出高效性能，具有直接的样品衍生化和对稳定的3-MPA衍生物的简单分析。
