Gozdowska Magdalena, Kulczykowska Ewa
Department of Genetics and Marine Biotechnology, Institute of Oceanology of Polish Academy of Sciences, Powstańców Warszawy 55 Str, 81-712 Sopot, Poland.
J Chromatogr B Analyt Technol Biomed Life Sci. 2004 Aug 5;807(2):229-33. doi: 10.1016/j.jchromb.2004.04.014.
A new HPLC assay for plasma arginine-vasotocin (AVT) and isotocin (IT) determination based on fluorescence detection preceded by combination of solid-phase extraction (SPE) and fluorescence derivatization is presented. Plasma samples retained on solid support were purified and then derivatized by the fluorescent compound 4-fluoro-7-nitro-2,1,3-benzoxadiazole (NBD-F). The peptide derivatives were eluted from cartridges, pre-concentrated and analyzed by HPLC system with fluorescent detection. The separation was carried out on a reversed-phase column with solvent gradient system. The assay was linear in the range 15-220 pmol ml(-1) for AVT r2=0.998 and 10-220 pmol ml(-1) for IT r2=0.996. The detection limits for AVT and IT were 0.8 and 0.5 pmol ml(-1) (3:1, signal-to-noise), respectively. The recoveries of derivatized hormones were in the range 89-93%. Both of the inter- and intra-day assay precision were below 5.5 and 9% for AVT and IT, respectively. The assay should be also applicable to plasma and tissue samples from other animals with only minor modification.
本文介绍了一种基于荧光检测的血浆精氨酸血管催产素（AVT）和异催产素（IT）的高效液相色谱（HPLC）检测方法，该方法先进行固相萃取（SPE）和荧光衍生化。保留在固相载体上的血浆样品经过纯化，然后用荧光化合物4-氟-7-硝基-2,1,3-苯并恶二唑（NBD-F）进行衍生化。肽衍生物从柱中洗脱，预浓缩后通过带有荧光检测的HPLC系统进行分析。分离在反相柱上采用溶剂梯度系统进行。该检测方法对AVT的线性范围为15 - 220 pmol ml(-1)，r2 = 0.998；对IT的线性范围为10 - 220 pmol ml(-1)，r = 0.996。AVT和IT的检测限分别为0.8和0.5 pmol ml(-1)（信噪比为3:1）。衍生化激素的回收率在89 - 93%范围内。AVT和IT的日间和日内检测精密度分别低于5.5%和9%。该检测方法只需进行少量修改，也应适用于其他动物的血浆和组织样品。
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