Ercal N, Yang P, Aykin N
Department of Chemistry, University of Missouri-Rolla, MO 65409-0010, USA.
J Chromatogr B Biomed Sci Appl. 2001 Apr 5;753(2):287-92. doi: 10.1016/s0378-4347(00)00560-0.
The importance of thiols has stimulated the development of a number of methods for determining glutathione and other biologically significant thiols. Methods that are currently available, however have some limitations, such as being time consuming and complex. In the present study, a new high-performance liquid chromatography (HPLC) method for determining biological thiols was developed by using 9-Acetoxy-2-(4-(2,5-dihydro-2,5-dioxo-1H-pyrrol-1-yl)phenyl)-3-oxo-3H-naphtho[2,1-b]pyran (ThioGlo3) as a derivatizing agent. ThioGlo reacts selectively and rapidly with the thiols to yield fluorescent adducts which can be detected fluorimetrically (lambda(ex) = 365 nm, lambda(em) = 445 nm). The within-run coefficient of variation for glutathione (GSH) by this method ranges from 1.08 to 2.94% whereas the between-run coefficient of variation for GSH is 4.31-8.61%. For GSH, the detection limit is around 50 fmol and the GSH derivatives remain stable for 1 month, if kept at 4 degrees C. Results for GSSG and cysteine are also included. The ThioGlo method is compared to our previous method in which N-(1-pyrenyl)maleimide (NPM) is used to derivatize thiol-containing compounds. The present method offers various advantages over the currently accepted techniques, including speed and sensitivity.
硫醇的重要性推动了多种测定谷胱甘肽及其他具有生物学意义的硫醇的方法的发展。然而，目前可用的方法存在一些局限性，比如耗时且复杂。在本研究中，开发了一种新的高效液相色谱（HPLC）法来测定生物硫醇，该方法使用9-乙酰氧基-2-(4-(2,5-二氢-2,5-二氧代-1H-吡咯-1-基)苯基)-3-氧代-3H-萘并[2,1-b]吡喃（硫代荧光素3，ThioGlo3）作为衍生剂。硫代荧光素与硫醇选择性且快速地反应，生成可通过荧光法检测的荧光加合物（激发波长λ(ex)=365nm，发射波长λ(em)=445nm）。用该方法测定谷胱甘肽（GSH）时，批内变异系数在1.08%至2.94%之间，而GSH的批间变异系数为4.31% - 8.61%。对于GSH，检测限约为50飞摩尔，若保存在4℃，GSH衍生物可稳定保存1个月。还包括了氧化型谷胱甘肽（GSSG）和半胱氨酸的测定结果。将硫代荧光素法与我们之前使用N-(1-芘基)马来酰亚胺（NPM）衍生含硫醇化合物的方法进行了比较。本方法相对于目前公认的技术具有多种优势，包括速度和灵敏度。
