柱前手性荧光衍生反相高效液相色谱法分离甲状腺素对映体

以R(-)-4-N,N-二甲基磺酰胺-7-(3-异氰酸吡咯烷)-2,1,3-苯并氧杂咪唑（R(-)-DBD-PyNCS）为手性荧光衍生化试剂,成功地拆分了甲状腺素对映体D,L-四碘甲状腺原氨酸（T4）和L-三碘甲状腺原氨酸(T3)。在反应温度为40 ℃、反应时间为20 min时,R(-)-DBD-PyNCS在碱性介质中可与甲状腺素对映体生成稳定的非对映体衍生物。该衍生物在以乙腈-水-醋酸(体积比为60∶40∶1)为流动相,流速为1.0 mL/min，色谱柱为Intersil-ODS-3 C18柱(150 mm×4.6 mm,5 μm)的色谱条件下得到了充分的分离。采用荧光检测器在激发波长460 nm、发射波长550 nm下检测。D,L-T4和L-T3分别在0.016～0.30 μg/μL和0.0067～0.22 μg/μL范围内,峰面积与浓度呈良好的线性关系(r＞0.999)。D,L-T4和L-T3的最低检出限分别为0.02 μg/mL和0.85 μg/mL(S/N=3)。在D-T4、L-T4、L-T3质量浓度分别为0.10 μg/μL下测得峰面积的相对标准偏差分别为3.40%，1.63%，3.30%(n=7)。该方法成功地应用于甲状腺片中T4和T3的含量测定。

Resolution of thyroxine hormone enantiomers by precolumn derivatization with a fluorescent chiral reagent using reversed-phase high performance liquid chromatography

A highly fluorescent chiral tagging reagent, R(-)-4-(N,N-dimethylaminosulfonyl)-7-(3-isothiocy-anatopyrrolidino)-2,1,3-benzoxadiazole （R(-)-DBD-PyNCS）, was employed for the enantiomer separation of thyroxine hormone, D,L-3,5,3′,5′-tetraiodothyronine （T4） and L-3,5,3′-triiodothyronine （T3）. The reaction of R(-)-DBD-PyNCS with the thyroxine enantiomers was carried out at 40 ℃ for 20 min under a basic medium surrounding to yield the corresponding pair of diastereomers. No racemization occurs during the tagging reaction under the optimized conditions. Various experimental parameters for the derivatization reaction including the concentration of tagging reagent, reaction temperature and reaction time have been studied in order to get the highest yield of T4/T3 derivatives. The structures of T4/T3 derivatives were identified based on high performance liquid chromatography-mass spectrometry （HPLC-MS） measurement in negative mode. The efficient separation of derivatives have been achieved by isocratic elution with a water-acetonitrile mobile phase containing 1% acetic acid in a reversed-phase column utilizing a conventional fluorescence detector. The calibration curves of L-T3, D,L-T4 were linear over the concentration ranges of 0.0067-0.22 μg/μL and 0.016-0.30 μg/μL, respectively. The limits of detection (S/N=3) for L-T3 and D,L-T4 were 0.85 μg/mL and 0.02 μg/mL, respectively. The proposed method has been successfully applied to the determination of T3 and T4 in clinical pharmaceutics.